Compositions and kits pertaining to analyte determination

ABSTRACT

This invention pertains to methods, mixtures, kits and/or compositions for the determination of analytes by mass analysis using unique labeling reagents or sets of unique labeling reagents. The labeling reagents can be isomeric or isobaric and can be used to produce mixtures suitable for multiplex analysis of the labeled analytes.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. Ser. No. 10/______entitled:“Methods And Mixtures Pertaining To Analyte Determination” and filed onthis Jan. 27^(th), 2004, incorporated herein by reference. Thisapplication is related to U.S. Ser. No. 10/______entitled: “Methods AndMixtures Pertaining To Analyte Determination Using ElectrophilicLabeling Reagents” and filed on this Jan. 27^(th), 2004, incorporatedherein by reference. This application claims the benefit of U.S.Provisional Patent Application Serial No. 60/443,612, filed on Jan. 30,2003, incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention pertains to the field of analyte determination bymass analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0003]FIG. 1A illustrates the reaction of an analyte with two differentisobaric labeling reagents (e.g. compounds I and II).

[0004]FIG. 1B illustrates the fragmentation of the labeled analyteillustrated in FIG. 1A to thereby produce reporter moieties (e.g.compounds VII and VIII as signature ions) of different masses from theisobarically labeled analytes.

[0005]FIG. 2 is an expansion plot of a mass spectrum of a labeledanalyte.

[0006]FIG. 3 is the complete mass spectrum obtained from a second massanalysis of the selected labeled analyte identified in the expansionplot of FIG. 2.

[0007]FIG. 4 is an expansion plot of a mass spectrum of the predominatey-ion daughter fragment ion of the analyte as determined in the secondmass analysis.

[0008]FIG. 5 is an expansion plot of a mass spectrum of the predominateb-ion daughter fragment ion of the analyte as determined in the secondmass analysis.

[0009]FIG. 6 is an expansion plot of a mass spectrum of two reporters(i.e. signature ions) as determined in the second mass analysis.

[0010]FIG. 7 is a plot of observed vs. predicted ratios of reportersdetermined by a second mass analysis for various mixtures of a labeledpeptide, each peptide of the mixture comprising one of two differentreporters.

[0011]FIG. 8 is an illustration of two sets of isobaric labelingreagents wherein the same isotopes (compounds X-XIII) and differentisotopes (compounds XV-XVIII) are used within the set to thereby achievereporter/linker moieties of the same gross mass but each with a reportermoiety of a different gross mass within the set.

[0012]FIGS. 9A and 9B are an illustration of synthetic routes toisotopically labeled piperazine labeling reagents from basic startingmaterials. The route can also be used to prepare non-isotopicallylabeled piperazine reagents wherein non-isotopically labeled startingmaterials are used.

[0013]FIG. 10 is an illustration of a synthetic route to isotopicallylabeled and non-isotopically labeled N-alkyl piperazine labelingreagents from basic starting materials.

[0014]FIG. 11 is an illustration of a synthetic route to isotopicallylabeled and non-isotopically labeled N-alkyl piperazine labelingreagents from basic starting materials.

[0015]FIG. 12 is an illustration of a solid phase based synthetic routeto isotopically labeled and non-isotopically labeled piperazine labelingreagents from basic starting materials.

1. INTRODUCTION

[0016] This invention pertains to methods, mixtures, kits and/orcompositions for the determination of an analyte or analytes by massanalysis. An analyte can be any molecule of interest. Non-limitingexamples of analytes include, but are not limited to, proteins,peptides, nucleic acids, carbohydrates, lipids, steroids and smallmolecules of less than 1500 daltons.

[0017] Analytes can be labeled by reaction of the analyte with alabeling reagent of the formula: RP-X-LK-Y-RG, or a salt thereof,wherein RG is a reactive group that reacts with the analyte and RP, X,LK and Y are described in more detail below. A labeled analyte thereforecan have the general formula: RP-X-LK-Y-Analyte. Sets of isomeric orisobaric labeling reagents can be used to label the analytes of two ormore different samples wherein the labeling reagent can be different foreach different sample and wherein the labeling reagent can comprise aunique reporter, “RP”, that can be associated with the sample from whichthe labeled analyte originated.

[0018] Hence, information, such as the presence and/or amount of thereporter, can be correlated with the presence and/or amount (oftenexpressed as a concentration and/or quantity) of the analyte in a sampleeven from the analysis of a complex mixture of labeled analytes derivedby mixing the products of the labeling of different samples. Analysis ofsuch complex sample mixtures can be performed in a manner that allowsfor the determination of one or a plurality of analytes from the same orfrom multiple samples in a multiplex manner. Thus, the methods,mixtures, kits and/or compositions of this invention are particularlywell suited for the multiplex analysis of complex sample mixtures. Forexample, they can be used in proteomic analysis and/or genomic analysisas well as for correlation studies related to genomic and proteomicanalysis.

2. Definitions

[0019] For the purposes of interpreting of this specification, thefollowing definitions will apply and whenever appropriate, terms used inthe singular will also include the plural and vice versa:

[0020] a. As used herein, “analyte” refers to a molecule of interestthat may be determined. Non-limiting examples of analytes can include,but are not limited to, proteins, peptides, nucleic acids (both DNA orRNA), carbohydrates, lipids, steroids and/or other small molecules witha molecular weight of less than 1500 daltons. The source of the analyte,or the sample comprising the analyte, is not a limitation as it can comefrom any source. The analyte or analytes can be natural or synthetic.Non-limiting examples of sources for the analyte, or the samplecomprising the analyte, include but are not limited to cells or tissues,or cultures (or subcultures) thereof. Non-limiting examples of analytesources include, but are not limited to, crude or processed cell lysates(including whole cell lysates), body fluids, tissue extracts or cellextracts. Still other non-limiting examples of sources for the analyteinclude but are not limited to fractions from a separations process suchas a chromatographic separation or an electrophoretic separation. Bodyfluids include, but are not limited to, blood, urine, feces, spinalfluid, cerebral fluid, amniotic fluid, lymph fluid or a fluid from aglandular secretion. By processed cell lysate we mean that the celllysate is treated, in addition to the treatments needed to lyse thecell, to thereby perform additional processing of the collectedmaterial. For example, the sample can be a cell lysate comprising one ormore analytes that are peptides formed by treatment of the total proteincomponent of a crude cell lysate with a proteolytic enzyme to therebydigest precursor protein or proteins.

[0021] b. As used herein, “fragmentation” refers to the breaking of acovalent bond.

[0022] c. As used herein, “fragment” refers to a product offragmentation (noun) or the operation of causing fragmentation (verb).

[0023] d. It is well accepted that the mass of an atom or molecule canbe approximated, often to the nearest whole number atomic mass unit orthe nearest tenth or hundredth of an atomic mass unit. As used herein,“gross mass” refers to the absolute mass as well as to the approximatemass within a range where the use of isotopes of different atom typesare so close in mass that they are the functional equivalent for thepurpose of balancing the mass of the reporter and/or linker moieties (sothat the gross mass of the reporter/linker combination is the samewithin a set or kit of isobaric or isomeric labeling reagents) whetheror not the very small difference in mass of the different isotopes typesused can be detected.

[0024] For example, the common isotopes of oxygen have a gross mass of16.0 (actual mass 15.9949) and 18.0 (actual mass 17.9992), the commonisotopes of carbon have a gross mass of 12.0 (actual mass 12.00000) and13.0 (actual mass 13.00336) and the common isotopes of nitrogen have agross mass of 14.0 (actual mass 14.0031) and 15.0 (actual mass 15.0001).Whilst these values are approximate, one of skill in the art willappreciate that if one uses the ¹⁸O isotope in one reporter of a set,the additional 2 mass units (over the isotope of oxygen having a grossmass of 16.0) can, for example, be compensated for in a differentreporter of the set comprising ¹⁶O by incorporating, elsewhere in thereporter, two carbon ¹³C atoms, instead of two ¹²C atoms, two ¹⁵N atoms,instead of two ¹⁴N atoms or even one ¹³C atom and one ¹⁵N atom, insteadof a ¹²C and a ¹⁴N, to compensate for the ¹⁸O. In this way the twodifferent reporters of the set are the functional mass equivalent (i.e.have the same gross mass) since the very small actual differences inmass between the use of two ¹³C atoms (instead of two ¹²C atoms), two¹⁵N atoms (instead of two ¹⁴N atoms), one ¹³C and one ¹⁵N (instead of a¹²C and ¹⁴N) or one ¹⁸O atom (instead of one ¹⁶O atom), to therebyachieve an increase in mass of two Daltons, in all of the labels of theset or kit, is not an impediment to the nature of the analysis.

[0025] This can be illustrated with reference to FIG. 8. In FIG. 8, thereporter/linker combination of compound XVII (FIG. 8; chemical formula:C₅ ¹³CH₁₀ ¹⁵N₂O) has two ¹⁵N atoms and one ¹³C atom and a totaltheoretical mass of 129.138. By comparison, isobar XV (FIG. 8; chemicalformula C₅ ¹³CH₁₀N₂ ¹⁸O) has one ¹⁸O atom and one ¹³C atom and a totaltheoretical mass of 129.151. Compounds XVII and XV are isobars that arestructurally and chemically indistinguishable, except for heavy atomisotope content, although there is a slight absolute mass difference(mass 129.138 vs. mass 129.151 respectively). However, the gross mass ofcompounds XVII and XV is 129.1 for the purposes of this invention sincethis is not an impediment to the analysis whether or not the massspectrometer is sensitive enough to measure the small difference betweenthe absolute mass of isobars XVII and XV.

[0026] From FIG. 8, it is clear that the distribution of the same heavyatom isotopes within a structure is not the only consideration for thecreation of sets of isomeric and/or isobaric labeling reagents. It ispossible to mix heavy atom isotope types to achieve isomers or isobarsof a desired gross mass. In this way, both the selection (combination)of heavy atom isotopes as well as their distribution is available forconsideration in the production of the isomeric and/or isobaric labelingreagents useful for embodiments of this invention.

[0027] e. As used herein, “isotopically enriched” refers to a compound(e.g. labeling reagent) that has been enriched synthetically with one ormore heavy atom isotopes (e.g. stable isotopes such as Deuterium, ¹³C,¹⁵N, ¹⁸O, ³⁷Cl or ⁸¹Br). Because isotopic enrichment is not 100%effective, there can be impurities of the compound that are of lesserstates of enrichment and these will have a lower mass. Likewise, becauseof over-enrichment (undesired enrichment) and because of naturalisotopic abundance, there can be impurities of greater mass.

[0028] f. As used herein, “labeling reagent” refers to a moiety suitableto mark an analyte for determination. The term label is synonymous withthe terms tag and mark and other equivalent terms and phrases. Forexample, a labeled analyte can also be referred to as a tagged analyteor a marked analyte. Accordingly the terms “label”, “tag”, “mark” andderivatives of these terms, are interchangeable and refer to a moietysuitable to mark, or that has marked, an analyte for determination.

[0029] g. As used herein, “support”, “solid support” or “solid carrier”means any solid phase material upon which a labeling reagent can beimmobilized. Immobilization can, for example, be used to label analytesor be used to prepare a labeling reagent, whether or not the labelingoccurs on the support. Solid support encompasses terms such as “resin”,“synthesis support”, “solid phase”, “surface” “membrane” and/or“support”. A solid support can be composed of organic polymers such aspolystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled-pore-glass (CPG), or reverse-phase silica. The configurationof a solid support can be in the form of beads, spheres, particles,granules, a gel, a membrane or a surface. Surfaces can be planar,substantially planar, or non-planar. Solid supports can be porous ornon-porous, and can have swelling or non-swelling characteristics. Asolid support can be configured in the form of a well, depression orother container, vessel, feature or location. A plurality of solidsupports can be configured in an array at various locations, addressablefor robotic delivery of reagents, or by detection methods and/orinstruments.

[0030] h. As used herein, “natural isotopic abundance” refers to thelevel (or distribution) of one or more isotopes found in a compoundbased upon the natural prevalence of an isotope or isotopes in nature.For example, a natural compound obtained from living plant matter willtypically contain about 0.6% ¹³C.

3. General

[0031] The Reactive Group:

[0032] The reactive group “RG” of the labeling reagent or reagents usedin the method, mixture, kit and/or composition embodiments can be eitheran electrophile or a nucleophile that is capable of reacting with one ormore reactive analytes of a sample. The reactive group can bepreexisting or it can be prepared in-situ. In-situ preparation of thereactive group can proceed in the absence of the reactive analyte or itcan proceed in the presence of the reactive analyte. For example, acarboxylic acid group can be modified in-situ with water-solublecarbodiimide (e.g. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride; EDC) to thereby prepare an electrophilic group that canbe reacted with a nucleophile such as an amine group. In someembodiments, activation of the carboxylic acid group of a labelingreagent with EDC can be performed in the presence of an amine(nucleophile) containing analyte. In some embodiments, the amine(nucleophile) containing analyte can also be added after the initialreaction with EDC is performed. In other embodiments, the reactive groupcan be generated in-situ by the in-situ removal of a protecting group.Consequently, any existing or newly created reagent or reagents that caneffect the derivatization of analytes by the reaction of nucleophilesand/or electrophiles are contemplated by the method, mixture, kit and/orcomposition embodiments of this invention.

[0033] Where the reactive group of the labeling reagent is anelectrophile, it can react with a suitable nucleophilic group of theanalyte or analytes. Where the reactive group of the labeling reagent isa nucleophile, it can react with a suitable electrophilic group of theanalyte or analytes. Numerous pairs of suitable nucleophilic groups andelectrophilic groups are known and often used in the chemical andbiochemical arts. Non-limiting examples of reagents comprising suitablenucleophilic or electrophilic groups that can be coupled to analytes(e.g. such as proteins, peptides, nucleic acids, carbohydrates, lipids,steroids or other small molecules of less that 1500 daltons) to effecttheir derivatization, are described in the Pierce Life Science &Analytical Research Products Catalog & Handbook (a Perstorp BiotecCompany), Rockford, Ill. 61105, USA. Other suitable reagents are wellknown in the art and are commercially available from numerous othervendors such as Sigma-Aldrich.

[0034] The reactive group of a labeling reagent can be an amine reactivegroup. For example the amine reactive group can be an active ester.Active esters are well known in peptide synthesis and refer to certainesters that are easily reacted with the N-α amine of an amino acid underconditions commonly used in peptide synthesis. The amine reactive activeester can be an N-hydroxysuccinimidyl ester, aN-hydroxysulfosuccinimidyl ester, a pentafluorophenyl ester, a2-nitrophenyl ester, a 4-nitrophenyl ester, a 2,4-dinitrophenylester ora 2,4-dihalophenyl ester. For example, the alcohol or thiol group of anactive ester can have the formula:

[0035] wherein X is O or S, but preferably O. All of the foregoing beingalcohol or thiol groups known to form active esters in the field ofpeptide chemistry wherein said alcohol or thiol group is displaced bythe reaction of the N-α-amine of the amino acid with the carbonyl carbonof the ester. It should be apparent that the active ester (e.g.N-hydroxysuccinimidyl ester) of any suitable labelling/tagging reagentdescribed herein could be prepared using well-known procedures (See:Greg T. Hermanson(1996). “The Chemistry of Reactive Groups” in“Bioconjugate Techniques” Chapter 2 pages 137-165, Academic Press,(NewYork); also see: Innovation And Perspectives In Solid PhaseSynthesis, Editor: Roger Epton, SPCC (UK) Ltd, Birmingham, 1990).Methods for the formation of active esters of N-substituted piperazineacetic acids compounds that are representative examples of labellingreagents of the general formula: RP-X-LK-Y-RG, are described inco-pending and commonly owned U.S. patent application Ser. No.10/751,354, incorporated herein by reference.

[0036] In another embodiment, the reactive group of the labeling reagentcan be a mixed anhydride since mixed anhydrides are known to efficientlyreact with amine groups to thereby produce amide bonds.

[0037] The reactive group of a labeling reagent can be a thiol reactivegroup. For example, the thiol reactive group can be a malemide, an alkylhalide, an aryl halide of an α-halo-acyl. By halide or halo we meanatoms of fluorine, chlorine, bromine or iodine.

[0038] The reactive group of a labeling reagent can be a hydroxylreactive group. For example, the hydroxyl reactive group can be atrityl-halide or a silyl-halide reactive moiety. The trityl-halidereactive moieties can be substituted (e.g. Y-methoxytrityl,Y-dimethoxytrityl, Y-trimethoxytrityl, etc) or unsubstituted wherein Yis defined below. The silyl reactive moieties can be alkyl substitutedsilyl halides, such as Y-dimethylsilyl, Y-ditriethylsilyl,Y-dipropylsilyl, Y-diisopropylsilyl, etc.) wherein Y is defined below.

[0039] The reactive group of the labeling reagent can be a nucleophilesuch as an amine group, a hydroxyl group or a thiol group.

[0040] The Reporter Moiety:

[0041] The reporter moiety of the labeling reagent or reagents used inthe method, mixture, kit and/or composition embodiments is a group thathas a unique mass (or mass to charge ratio) that can be determined.Accordingly, each reporter of a set can have a unique gross mass.Different reporters can comprise one or more heavy atom isotopes toachieve their unique mass. For example, isotopes of carbon (¹²C, ¹³C and¹⁴C), nitrogen (¹⁴N and ¹⁵N), oxygen (¹⁶O and ¹⁸O) or hydrogen(hydrogen, deuterium and tritium) exist and can be used in thepreparation of a diverse group of reporter moieties. Examples of stableheavy atom isotopes include ¹³C, ¹⁵N, ¹⁸O and deuterium. These are notlimiting as other light and heavy atom isotopes can also be used in thereporter. Basic starting materials suitable for preparing reporterscomprising light and heavy atom isotopes are available from variouscommercial sources such as Cambridge Isotope Laboratories, Andover,Mass. (See: list or “basic starting materials” at www.isotope.com) andIsotec (a division of Sigma-Aldrich). Cambridge Isotope Laboratories andIsotec will also prepare desired compounds under custom synthesiscontracts. Id.

[0042] A unique reporter can be associated with a sample of interestthereby labeling one or multiple analytes of that sample with thereporter. In this way information about the reporter can be associatedwith information about one or all of the analytes of the sample.However, the reporter need not be physically linked to an analyte whenthe reporter is determined. Rather, the unique gross mass of thereporter can, for example, be determined in a second mass analysis of atandem mass analyzer, after ions of the labeled analyte are fragmentedto thereby produce daughter fragment ions and detectable reporters. Thedetermined reporter can be used to identify the sample from which adetermined analyte originated. Further, the amount of the uniquereporter, either relative to the amount of other reporters or relativeto a calibration standard (e.g. an analyte labeled with a specificreporter), can be used to determine the relative or absolute amount(often expressed as a concentration and/or quantity) of analyte in thesample or samples. Therefore information, such as the amount of one ormore analytes in a particular sample, can be associated with thereporter moiety that is used to label each particular sample. Where theidentity of the analyte or analytes is also determined, that informationcan be correlated with information pertaining to the different reportersto thereby facilitate the determination of the identity and amount ofeach labeled analyte in one or a plurality of samples.

[0043] The reporter either comprises a fixed charge or is capable ofbecoming ionized. Because the reporter either comprises a fixed chargeor is capable of being ionized, the labeling reagent might be isolatedor used to label the reactive analyte in a salt or zwitterionic form.Ionization of the reporter facilitates its determination in a massspectrometer. Accordingly, the reporter can be determined as a ion,sometimes referred to as a signature ion. When ionized, the reporter cancomprise one or more net positive or negative charges. Thus, thereporter can comprise one or more acidic groups or basic groups sincesuch groups can be easily ionized in a mass spectrometer. For example,the reporter can comprise one or more basic nitrogen atoms (positivecharge) or one or more ionizable acidic groups such as a carboxylic acidgroup, sulfonic acid group or phosphoric acid group (negative charge).Non-limiting examples of reporters comprising a basic nitrogen include,substituted or unsubstituted, morpholines, piperidines or piperazines.

[0044] The reporter can be a 5, 6 or 7 membered heterocyclic ringcomprising a ring nitrogen atom that is N-alkylated with a substitutedor unsubstituted acetic acid moiety to which the analyte is linkedthrough the carbonyl carbon of the N-alkyl acetic acid moiety, whereineach different label comprises one or more heavy atom isotopes. Theheterocyclic ring can be substituted or unsubstituted. The heterocyclicring can be aliphatic or aromatic. Possible substituents of theheterocylic moiety include alkyl, alkoxy and aryl groups. Thesubstituents can comprise protected or unprotected groups, such asamine, hydroxyl or thiol groups, suitable for linking the analyte to asupport. The heterocyclic ring can comprise additional heteroatoms suchas one or more nitrogen, oxygen or sulfur atoms.

[0045] The reporter can be selected so that it does not substantiallysub-fragment under conditions typical for the analysis of the analyte.The reporter can be chosen so that it does not substantiallysub-fragment under conditions of dissociative energy applied to causefragmentation of both bonds X and Y of at least a portion of selectedions of a labeled analyte in a mass spectrometer. By “does notsubstantially sub-fragment” we mean that fragments of the reporter aredifficult or impossible to detect above background noise when applied tothe successful analysis of the analyte of interest. The gross mass of areporter can be intentionally selected to be different as compared withthe mass of the analyte sought to be determined or any of the expectedfragments of the analyte. For example, where proteins or peptides arethe analytes, the reporter's gross mass can be chosen to be different ascompared with any naturally occurring amino acid or peptide, or expectedfragments thereof. This can facilitate analyte determination since,depending on the analyte, the lack of any possible components of thesample having the same coincident mass can add confidence to the resultof any analysis.

[0046] The reporter can be a small molecule that is non-polymeric. Thereporter does not have to be a biopolymer (e.g. a peptide, a protein ora nucleic acid) or a component of a biopolymer (e.g. an amino acid, anucleoside or a nucleotide). The gross mass of a reporter can be lessthan 250 Daltons. Such a small molecule can be easily determined in thesecond mass analysis, free from other components of the sample havingthe same coincident mass in the first mass analysis. In this context,the second mass analysis can be performed, typically in a tandem massspectrometer, on selected ions that are determined in the first massanalysis. Because ions of a particular mass to charge ratio can bespecifically selected out of the first mass analysis for possiblefragmentation and further mass analysis, the non-selected ions from thefirst mass analysis are not carried forward to the second mass analysisand therefore do not contaminate the spectrum of the second massanalysis. Furthermore, the sensitivity of a mass spectrometer and thelinearity of the detector (for purposes of quantitation) can be quiterobust in this low mass range. Additionally, the present state of massspectrometer technology can allow for baseline mass resolution of lessthan one Dalton in this mass range (See for example: FIG. 6). Thesefactors may prove to be useful advancements to the state of the art.

[0047] The Linker Moiety:

[0048] The linker moiety of the labeling reagent or reagents used withthe method, mixture, kit and/or composition embodiments links thereporter to the analyte or the reporter to the reactive group dependingon whether or not a reaction with the analyte has occurred. The linkercan be selected to produce a neutral species when both bonds X and Y arefragmented (i.e. undergoes neutral loss upon fragmentation of both bondsX and Y). The linker can be a very small moiety such as a carbonyl orthiocarbonyl group. For example, the linker can comprise at least oneheavy atom isotope and comprise the formula:

[0049] wherein R¹ is the same or different and is an alkyl groupcomprising one to eight carbon atoms which may optionally contain aheteroatom or a substituted or unsubstituted aryl group wherein thecarbon atoms of the alkyl and aryl groups independently comprise linkedhydrogen, deuterium and/or fluorine atoms. The linker can be a largermoiety. The linker can be a polymer or a biopolymer. The linker can bedesigned to sub-fragment when subjected to dissociative energy levels;including sub-fragmentation to thereby produce only neutral fragments ofthe linker.

[0050] The linker moiety can comprise one or more heavy atom isotopessuch that its mass compensates for the difference in gross mass betweenthe reporters for each labeled analyte of a mixture or for the reagentsof set and/or kit. Moreover, the aggregate gross mass (i.e. the grossmass taken as a whole) of the reporter/linker combination can be thesame for each labeled analyte of a mixture or for the reagents of setand/or kit. More specifically, the linker moiety can compensate for thedifference in gross mass between reporters of labeled analytes fromdifferent samples wherein the unique gross mass of the reportercorrelates with the sample from which the labeled analyte originated andthe aggregate gross mass of the reporter/linker combination is the samefor each labeled analyte of a sample mixture regardless of the samplefrom which it originated. In this way, the gross mass of identicalanalytes in two or more different samples can have the same gross masswhen labeled and then mixed to produce a sample mixture.

[0051] For example, the labeled analytes, or the reagents of a setand/or kit for labeling the analytes, can be isomers or isobars. Thus,if ions of a particular mass to charge ratio (taken from the samplemixture) are selected (i.e. selected ions) in a mass spectrometer froman initial mass analysis of the sample mixture, identical analytes fromthe different samples that make up the sample mixture are represented inthe selected ions in proportion to their respective concentration and/orquantity in the sample mixture. Accordingly, the linker not only linksthe reporter to the analyte, it also can serve to compensate for thediffering masses of the unique reporter moieties to thereby harmonizethe gross mass of the reporter/linker combination in the labeledanalytes of the various samples.

[0052] Because the linker can act as a mass balance for the reporter inthe labeling reagents, such that the aggregate gross mass of thereporter/linker combination is the same for all reagents of a set orkit, the greater the number of atoms in the linker, the greater thepossible number of different isomeric/isobaric labeling reagents of aset and/or kit. Stated differently, generally the greater the number ofatoms that a linker comprises, the greater number of potentialreporter/linker combinations exist since isotopes can be substituted atmost any position in the linker to thereby produce isomers or isobars ofthe linker portion wherein the linker portion is used to offset thediffering masses of the reporter portion and thereby create a set ofreporter/linker isomers or isobars. Such diverse sets of labelingreagents are particularly well suited for multiplex analysis of analytesin the same and/or different samples.

[0053] The total number of labeling reagents of a set and/or kit can betwo, three, four, five, six, seven, eight, nine, ten or more. Thediversity of the labeling reagents of a set or kit is limited only bythe number of atoms of the reporter and linker moieties, the heavy atomisotopes available to substitute for the light isotopes and the varioussynthetic configurations in which the isotopes can be syntheticallyplaced. As suggested above however, numerous isotopically enriched basicstarting materials are readily available from manufacturers such asCambridge Isotope Laboratories and Isotec. Such isotopically enrichedbasic starting materials can be used in the synthetic processes used toproduce sets of isobaric and isomeric labeling reagents or be used toproduce the isotopically enriched starting materials that can be used inthe synthetic processes used to produce sets of isobaric and isomericlabeling reagents. Some examples of the preparation of isobaric labelingreagents suitable for use in a set of labeling reagents can be found inthe Examples section, below.

[0054] The Reporter/Linker Combination:

[0055] The labeling reagents described herein comprise reporters andlinkers that are linked through the bond X. As described above, thereporter/linker combination can be identical in gross mass for eachmember of a set and/or kit of labeling reagents. Moreover, bond X of thereporter/linker combination of the labeling reagents can be designed tofragment, in at least a portion of the selected ions, when subjected todissociative energy levels thereby releasing the reporter from theanalyte. Accordingly, the gross mass of the reporter (as a m/s ratio)and its intensity can be observed directly in MS/MS analysis.

[0056] The reporter/linker combination can comprise various combinationsof the same or different heavy atom isotopes amongst the variouslabeling reagents of a set or kit. In the scientific literature this hassometimes been referred to as coding or isotope coding. For example,Abersold et al. has disclosed the isotope coded affinity tag (ICAT; seeWO00/11208). In one respect, the reagents of Abersold et al. differ fromthe labeling reagents of this invention in that Abersold does not teachtwo or more same mass labeling reagents such as isomeric or isobariclabeling reagents.

[0057] Mass Spectrometers/Mass Spectrometry (MS):

[0058] The methods of this invention can be practiced using tandem massspectrometers and other mass spectrometers that have the ability toselect and fragment molecular ions. Tandem mass spectrometers (and to alesser degree single-stage mass spectrometers) have the ability toselect and fragment molecular ions according to their mass-to-charge(m/z) ratio, and then record the resulting fragment (daughter) ionspectra. More specifically, daughter fragment ion spectra can begenerated by subjecting selected ions to dissociative energy levels(e.g. collision-induced dissociation (CID)). For example, ionscorresponding to labeled peptides of a particular m/z ratio can beselected from a first mass analysis, fragmented and reanalyzed in asecond mass analysis. Representative instruments that can perform suchtandem mass analysis include, but are not limited to, magneticfour-sector, tandem time-of-flight, triple quadrupole, ion-trap, andhybrid quadrupole time-of-flight (Q-TOF) mass spectrometers.

[0059] These types of mass spectrometers may be used in conjunction witha variety of ionization sources, including, but not limited to,electrospray ionization (ESI) and matrix-assisted laser desorptionionization (MALDI). Ionization sources can be used to generate chargedspecies for the first mass analysis where the analytes do not alreadypossess a fixed charge. Additional mass spectrometry instruments andfragmentation methods include post-source decay in MALDI-MS instrumentsand high-energy CID using MALDI-TOF(time of flight)-TOF MS. For a recentreview of tandem mass spectrometers please see: R. Aebersold and D.Goodlett, Mass Spectrometry in Proteomics. Chem. Rev. 101: 269-295(2001). Also see U.S. Pat. No. 6,319,476, herein incorporated byreference, for a discussion of TOF-TOF mass analysis techniques.

[0060] Fragmentation by Dissociative Energy Levels:

[0061] It is well accepted that bonds can fragment as a result of theprocesses occurring in a mass spectrometer. Moreover, bond fragmentationcan be induced in a mass spectrometer by subjecting ions to dissociativeenergy levels. For example, the dissociative energy levels can beproduced in a mass spectrometer by collision-induced dissociation (CID).Those of ordinary skill in the art of mass spectrometry will appreciatethat other exemplary techniques for imposing dissociative energy levelsthat cause fragmentation include, but are not limited to, photodissociation, electron capture and surface induced dissociation.

[0062] The process of fragmenting bonds by collision-induceddissociation involves increasing the kinetic energy state of selectedions, through collision with an inert gas, to a point where bondfragmentation occurs. For example, kinetic energy can be transferred bycollision with an inert gas (such as nitrogen, helium or argon) in acollision cell. The amount of kinetic energy that can be transferred tothe ions is proportional to the number of gas molecules that are allowedto enter the collision cell. When more gas molecules are present, agreater amount of kinetic energy can be transferred to the selectedions, and less kinetic energy is transferred when there are fewer gasmolecules present.

[0063] It is therefore clear that the dissociative energy level in amass spectrometer can be controlled. It is also well accepted thatcertain bonds are more labile than other bonds. The lability of thebonds in an analyte or the reporter/linker moiety depends upon thenature of the analyte or the reporter/linker moiety. Accordingly, thedissociative energy levels can be adjusted so that the analytes and/orthe labels (e.g. the reporter/linker combinations) can be fragmented ina manner that is determinable. One of skill in the art will appreciatehow to make such routine adjustments to the components of a massspectrometer to thereby achieve the appropriate level of dissociativeenergy to thereby fragment at least a portion of ions of labeledanalytes into ionized reporter moieties and daughter fragment ions.

[0064] For example, dissociative energy can be applied to ions that areselected/isolated from the first mass analysis. In a tandem massspectrometer, the extracted ions can be subjected to dissociative energylevels and then transferred to a second mass analyzer. The selected ionscan have a selected mass to charge ratio. The mass to charge ratio canbe within a range of mass to charge ratios depending upon thecharacteristics of the mass spectrometer. When collision induceddissociation is used, the ions can be transferred from the first to thesecond mass analyzer by passing them through a collision cell where thedissociative energy can be applied to thereby produce fragment ions. Forexample the ions sent to the second mass analyzer for analysis caninclude some, or a portion, of the remaining (unfragmented) selectedions, as well as reporter ions (signature ions) and daughter fragmentions of the labeled analyte.

[0065] Analyte Determination by Computer Assisted Database Analysis:

[0066] In some embodiments, analytes can be determined based upondaughter-ion fragmentation patterns that are analyzed bycomputer-assisted comparison with the spectra of known or “theoretical”analytes. For example, the daughter fragment ion spectrum of a peptideion fragmented under conditions of low energy CID can be considered thesum of many discrete fragmentation events. The common nomenclaturedifferentiates daughter fragment ions according to the amide bond thatbreaks and the peptide fragment that retains charge following bondfission. Charge-retention on the N-terminal side of the fissile amidebond results in the formation of a b-type ion. If the charge remains onthe C-terminal side of the broken amide bond, then the fragment ion isreferred to as a y-type ion. In addition to b- and y-type ions, the CIDmass spectrum may contain other diagnostic fragment ions (daughterfragment ions). These include ions generated by neutral loss of ammonia(−17 amu) from glutamine, lysine and arginine or the loss of water (−18amu) from hydroxyl-containing amino acids such as serine and threonine.Certain amino acids have been observed to fragment more readily underconditions of low-energy CID than others. This is particularly apparentfor peptides containing proline or aspartic acid residues, and even moreso at aspartyl-proline bonds (Mak, M. et al., Rapid Commun. MassSpectrom., 12: 837-842) (1998). Accordingly, the peptide bond of a Z-prodimer or Z-asp dimer, wherein Z is any natural amino acid, pro isproline and asp is aspartic acid, will tend to be more labile ascompared with the peptide bond between all other amino acid dimercombinations.

[0067] For peptide and protein samples therefore, low-energy CID spectracontain redundant sequence-specific information in overlapping b- andy-series ions, internal fragment ions from the same peptide, andimmonium and other neutral-loss ions. Interpreting such CID spectra toassemble the amino acid sequence of the parent peptide de novo ischallenging and time-consuming. The most significant advances inidentifying peptide sequences have been the development of computeralgorithms that correlate peptide CID spectra with peptide sequencesthat already exist in protein and DNA sequence databases. Suchapproaches are exemplified by programs such as SEQUEST (Eng, J. et al.J. Am. Soc. Mass Spectrom., 5: 976-989 (1994)) and MASCOT (Perkins, D.et al. Electrophoresis, 20: 3551-3567 (1999)).

[0068] In brief, experimental peptide CID spectra (MS/MS spectra) arematched or correlated with ‘theoretical’ daughter fragment ion spectracomputationally generated from peptide sequences obtained from proteinor genome sequence databases. The match or correlation is based upon thesimilarities between the expected mass and the observed mass of thedaughter fragment ions in MS/MS mode. The potential match or correlationis scored according to how well the experimental and ‘theoretical’fragment patterns coincide. The constraints on databases searching for agiven peptide amino acid sequence are so discriminating that a singlepeptide CID spectrum can be adequate for identifying any given proteinin a whole-genome or expressed sequence tag (EST) database. For otherreviews please see: Yates, J. R. Trends, Genetics, 16: 5-8 (2000) andYates, J. R., Electrophoresis 19: 893-900 (1998).

[0069] Accordingly, daughter fragment ion analysis of MS/MS spectra canbe used not only to determine the analyte of a labeled analyte, it canalso be used to determine analytes from which the determined analyteoriginated. For example, identification of a peptide in the MS/MSanalysis can be can be used to determine the protein from which thepeptide was cleaved as a consequence of an enzymatic digestion of theprotein. It is envisioned that such analysis can be applied to otheranalytes, such as nucleic acids.

[0070] Bonds X and Y:

[0071] X is a bond between an atom of the reporter and an atom of thelinker. Y is a bond between an atom of the linker and an atom of eitherthe reactive group or, if the labeling reagent has been reacted with areactive analyte, the analyte. Bonds X and Y of the various labelingreagents (i.e. RP-X-LK-Y-RG) that can be used in the embodiments of thisinvention can fragment, in at least a portion of selected ions, whensubjected to dissociative energy levels. Therefore, the dissociativeenergy level can be adjusted in a mass spectrometer so that both bonds Xand Y fragment in at least a portion of the selected ions of the labeledanalytes (i.e. RP-X-LK-Y-Analyte). Fragmentation of bond X releases thereporter from the analyte so that the reporter can be determinedindependently from the analyte. Fragmentation of bond Y releases thereporter/linker combination from the analyte, or the linker from theanalyte, depending on whether or not bond X has already been fragmented.Bond Y can be more labile than bond X. Bond X can be more labile thanbond Y. Bonds X and Y can be of the same relative lability.

[0072] When the analyte of interest is a protein or peptide, therelative lability of bonds X and Y can be adjusted with regard to anamide (peptide) bond. Bond X, bond Y or both bonds X and Y can be more,equal or less labile as compared with a typical amide (peptide) bond.For example, under conditions of dissociative energy, bond X and/or bondY can be less prone to fragmentation as compared with the peptide bondof a Z-pro dimer or Z-asp dimer, wherein Z is any natural amino acid,pro is proline and asp is aspartic acid. In some embodiments, bonds Xand Y will fragment with approximately the same level of dissociativeenergy as a typical amide bond. In some embodiments, bonds X and Y willfragment at a greater level of dissociative energy as compared with atypical amide bond.

[0073] Bonds X and Y can also exist such that fragmentation of bond Yresults in the fragmentation of bond X, and vice versa. In this way,both bonds X and Y can fragment essentially simultaneously such that nosubstantial amount of analyte, or daughter fragment ion thereof,comprises a partial label in the second mass analysis. By “substantialamount of analyte” we mean that less than 25%, and preferably less than10%, partially labeled analyte can be determined in the MS/MS spectrum.

[0074] Because there can be a clear demarcation between labeled andunlabeled fragments of the analyte in the spectra of the second massanalysis (MS/MS), this feature can simplify the identification of theanalytes from computer assisted analysis of the daughter fragment ionspectra. Moreover, because the fragment ions of analytes can, in someembodiments, be either fully labeled or unlabeled (but not partiallylabeled) with the reporter/linker moiety, there can be little or noscatter in the masses of the daughter fragment ions caused by isotopicdistribution across fractured bonds such as would be the case whereisotopes were present on each side of a single labile bond of apartially labeled analyte routinely determined in the second massanalysis.

[0075] Sample Processing:

[0076] In certain embodiments of this invention, a sample can beprocessed prior to, as well as after, labeling of the analytes. Theprocessing can facilitate the labeling of the analytes. The processingcan facilitate the analysis of the sample components. The processing cansimplify the handling of the samples. The processing can facilitate twoor more of the foregoing.

[0077] For example, a sample can be treated with an enzyme. The enzymecan be a protease (to degrade proteins and peptides), a nuclease (todegrade nucleic acids) or some other enzyme. The enzyme can be chosen tohave a very predictable degradation pattern. Two or more proteasesand/or two or more nuclease enzymes may also be used together, or withother enzymes, to thereby degrade sample components.

[0078] For example, the proteolytic enzyme trypsin is a serine proteasethat cleaves peptide bonds between lysine or arginine and an unspecificamino acid to thereby produce peptides that comprise an amine terminus(N-terminus) and lysine or arginine carboxyl terminal amino acid(C-terminus). In this way the peptides from the cleavage of the proteinare predictable and their presence and/or quantity, in a sample from atrypsin digest, can be indicative of the presence and/or quantity of theprotein of their origin. Moreover, the free amine termini of a peptidecan be a good nucleophile that facilitates its labeling. Other exemplaryproteolytic enzymes include papain, pepsin, ArgC, LysC, V8 protease,AspN, pronase, chymotrypsin and carboxypeptidease C.

[0079] For example, a protein (e.g. protein Z) might produce threepeptides (e.g. peptides B, C and D) when digested with a protease suchas trypsin. Accordingly, a sample that has been digested with aproteolytic enzyme, such as trypsin, and that when analyzed is confirmedto contain peptides B, C and D, can be said to have originally comprisedthe protein Z. The quantity of peptides B, C and D will also correlatewith the quantity of protein Z in the sample that was digested. In thisway, any determination of the identity and/or quantify of one or more ofpeptides B, C and D in a sample (or a fraction thereof, can be used toidentify and/or quantify protein Z in the original sample (or a fractionthereof).

[0080] Because activity of the enzymes is predictable, the sequence ofpeptides that are produced from degradation of a protein of knownsequence can be predicted. With this information, “theoretical” peptideinformation can be generated. A determination of the “theoretical”peptide fragments in computer assisted analysis of daughter fragmentions (as described above) from mass spectrometry analysis of an actualsample can therefore be used to determine one or more peptides orproteins in one or more unknown samples.

[0081] Separation of the Sample Mixture:

[0082] In some embodiments the processing of a sample or sample mixtureof labeled analytes can involve separation. For example, a samplemixture comprising differentially labeled analytes from differentsamples can be prepared. By differentially labeled we mean that each ofthe labels comprises a unique property that can be identified (e.g.comprises a unique reporter moiety that produces a unique “signatureion” in MS/MS analysis). In order to analyze the sample mixture,components of the sample mixture can be separated and mass analysisperformed on only a fraction of the sample mixture. In this way, thecomplexity of the analysis can be substantially reduced since separatedanalytes can be individually analyzed for mass thereby increasing thesensitivity of the analysis process. Of course the analysis can berepeated one or more time on one or more additional fractions of thesample mixture to thereby allow for the analysis of all fractions of thesample mixture.

[0083] Separation conditions under which identical analytes that aredifferentially labeled co-elute at a concentration, or in a quantity,that is in proportion to their abundance in the sample mixture can beused to determine the amount of each labeled analyte in each of thesamples that comprise the sample mixture provided that the amount ofeach sample added to the sample mixture is known. Accordingly, in someembodiments, separation of the sample mixture can simplify the analysiswhilst maintaining the correlation between signals determined in themass analysis (e.g. MS/MS analysis) with the amount of the differentlylabeled analytes in the sample mixture.

[0084] The separation can be performed by chromatography. For example,liquid chromatography/mass spectrometry (LC/MS) can be used to effectsuch a sample separation and mass analysis. Moreover, anychromatographic separation process suitable to separate the analytes ofinterest can be used. For example, the chromatographic separation can benormal phase chromatography, reversed-phase chromatography, ion-exchangechromatography, size exclusion chromatography or affinitychromatorgraphy.

[0085] The separation can be performed electrophoretically. Non-limitingexamples of electrophoretic separations techniques that can be usedinclude, but are not limited to, 1D electrophoretic separation, 2Delectrophoretic separation and/or capillary electrophoretic separation.

[0086] An isobaric labeling reagent or a set of reagents can be used tolabel the analytes of a sample. Isobaric labeling reagents areparticularly useful when a separation step is performed because theisobaric labels of a set of labeling reagents are structurally andchemically indistinguishable (and can be indistinguishable by gross massuntil fragmentation removes the reporter from the analyte). Thus, allanalytes of identical composition that are labeled with differentisobaric labels can chromatograph in exactly the same manner (i.e.co-elute). Because they are structurally and chemicallyindistinguishable, the eluent from the separation process can comprisean amount of each isobarically labeled analyte that is in proportion tothe amount of that labeled analyte in the sample mixture. Furthermore,from the knowledge of how the sample mixture was prepared (portions ofsamples, an other optional components (e.g. calibration standards) addedto prepare the sample mixture), it is possible to relate the amount oflabeled analyte in the sample mixture back to the amount of that labeledanalyte in the sample from which it originated.

[0087] The labeling reagents can also be isomeric. Although isomers cansometimes be chromatographically separated, there are circumstances,that are condition dependent, where the separation process can beoperated to co-elute all of the identical analytes that aredifferentially labeled wherein the amount of all of the labeled analytesexist in the eluent in proportion to their concentration and/or quantityin the sample mixture.

[0088] As used herein, isobars differ from isomers in that isobars arestructurally and chemically indistinguishable compounds (except forisotopic content and/or distribution) of the same nominal gross mass(See for example, FIG. 1) whereas isomers are structurally and/orchemically distinguishable compounds of the same nominal gross mass.

[0089] Relative and Absolute Quantitation of Analytes:

[0090] In some embodiments, the relative quantitation of differentiallylabeled identical analytes of a sample mixture is possible. Relativequantitation of differentially labeled identical analytes is possible bycomparison of the relative amounts of reporter (e.g. area or height ofthe peak reported) that are determined in the second mass analysis for aselected, labeled analyte observed in a first mass analysis. Putdifferently, where each reporter can be correlated with information fora particular sample used to produce a sample mixture, the relativeamount of that reporter, with respect to other reporters observed in thesecond mass analysis, is the relative amount of that analyte in thesample mixture. Where components combined to form the sample mixture isknown, the relative amount of the analyte in each sample used to preparethe sample mixture can be back calculated based upon the relativeamounts of reporter observed for the ions of the labeled analyteselected from the first mass analysis. This process can be repeated forall of the different labeled analytes observed in the first massanalysis. In this way, the relative amount (often expressed in terms ofconcentration and/or quantity) of each reactive analyte, in each of thedifferent samples used to produce the sample mixture, can be determined.

[0091] In other embodiments, absolute quantitation of analytes can bedetermined. For these embodiments, a known amount of one or moredifferentially labeled analytes (the calibration standard or calibrationstandards) can be added to the sample mixture. The calibration standardcan be an expected analyte that is labeled with an isomeric or isobariclabel of the set of labels used to label the analytes of the samplemixture provided that the reporter for the calibration standard isunique as compared with any of the samples used to form the samplemixture. Once the relative amount of reporter for the calibrationstandard, or standards, is determined with relation to the relativeamounts of the reporter for the differentially labeled analytes of thesample mixture, it is possible to calculate the absolute amount (oftenexpressed in concentration and/or quantity) of all of the differentiallylabeled analytes in the sample mixture. In this way, the absolute amountof each differentially labeled analyte (for which there is a calibrationstandard in the sample from which the analyte originated) can also bedetermined based upon the knowledge of how the sample mixture wasprepared.

[0092] Notwithstanding the foregoing, corrections to the intensity ofthe reporters (signature ions) can be made, as appropriate, for anynaturally occurring, or artificially created, isotopic abundance withinthe reporters. An example of such a correction can be found in Example3. A more sophisticated example of these types of corrections can alsobe found in copending and co-owned U.S. Provisional Patent ApplicationSerial No. 60/524,844, entitled: “Method and Apparatus ForDe-Convoluting A Convoluted Spectrum”, filed on Nov. 26, 2003. The morecare taken to accurately quantify the intensity of each reporter, themore accurate will be the relative and absolute quantification of theanalytes in the original samples.

[0093] Proteomic Analysis:

[0094] The methods, mixtures, kits and/or compositions of this inventioncan be used for complex analysis because samples can be multiplexed,analyzed and reanalyzed in a rapid and repetitive manner using massanalysis techniques. For example, sample mixtures can be analyzed forthe amount of individual analytes in one or more samples. The amount(often expressed in concentration and/or quantity) of those analytes canbe determined for the samples from which the sample mixture wascomprised. Because the sample processing and mass analyses can beperformed rapidly, these methods can be repeated numerous times so thatthe amount of many differentially labeled analytes of the sample mixturecan be determined with regard to their relative and/or absolute amountsin the sample from which the analyte originated.

[0095] One application where such a rapid multiplex analysis is usefulis in the area of proteomic analysis. Proteomics can be viewed as anexperimental approach to describe the information encoded in genomicsequences in terms of structure, function and regulation of biologicalprocesses. This may be achieved by systematic analysis of the totalprotein component expressed by a cell or tissue. Mass spectrometry, usedin combination with the method, mixture, kit and/or compositionembodiments of this invention is one possible tool for such globalprotein analysis.

[0096] For example, with a set of four isobaric labeling reagents, it ispossible to obtain four time points in an experiment to determine up ordown regulation of protein expression, for example, based upon responseof growing cells to a particular stimulant. It is also possible toperform fewer time points but to incorporate one or two controls. In allcases, up or down regulation of the protein expression, optionally withrespect to the controls, can be determined in a single multiplexexperiment. Moreover, because processing is performed in parallel theresults are directly comparable, since there is no risk that slightvariations in protocol may have affected the results.

4. Description of Various Embodiments of the Invention

[0097] A. Methods

[0098] According to the methods of this invention, the analyte to bedetermined is labeled. The labeled analyte, the analyte itself, one ormore fragments of the analyte and/or fragments of the label, can bedetermined by mass analysis. In some embodiments, methods of thisinvention can be used for the analysis of different analytes in the samesample as well as for the multiplex analysis of the same and/ordifferent analytes in two or more different samples. The two or moresamples can be mixed to form a sample mixture. In the multiplexanalysis, labeling reagents can be used to determine from which sampleof a sample mixture an analyte originated. The absolute and/or relative(with respect to the same analyte in different samples) amount (oftenexpressed in concentration or quantity) of the analyte, in each of twoor more of the samples combined to form the sample mixture, can bedetermined. Moreover, the mass analysis of fragments of the analyte(e.g. daughter fragment ions) can be used to identify the analyte and/orthe precursor to the analyte; such as where the precursor molecule tothe analyte was degraded.

[0099] One distinction of the described approach lies in the fact thatanalytes from different samples can be differentially isotopicallylabeled (i.e. isotopically coded) with unique labels that are chemicallyisomeric or isobaric (have equal mass) and that identify the sample fromwhich the analyte originated. The differentially labeled analytes arenot distinguished in MS mode of a mass spectrometer because they allhave identical (gross) mass to charge ratios. However, when subjected todissociative energy levels, such as through collision induceddissociation (CID), the labels can fragment to yield unique reportersthat can be resolved by mass (mass to charge ratio) in a massspectrometer. The relative amount of reporter observed in the massspectrum can correlate with the relative amount of a labeled analyte inthe sample mixture and, by implication, the amount of that analyte in asample from which it originated. Thus, the relative intensities of thereporters (i.e. signature ions) can be used to measure the relativeamount of an analyte or analytes in two or more different samples thatwere combined to form a sample mixture. From the reporter information,absolute amounts (often expressed as concentration and/or quantity) ofan analyte or analytes in two or more samples can be derived ifcalibration standards for the each analyte, for which absolutequantification is desired, are incorporated into the sample mixture.

[0100] For example, the analyte might be a peptide that resulted fromthe degradation of a protein using an enzymatic digestion reaction toprocess the sample. Protein degradation can be accomplished by treatmentof the sample with a proteolytic enzyme (e.g. trypsin, papain, pepsin,ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin orcarboxypeptidease C). By determination of the identity and amount of apeptide in a sample mixture and identifying the sample from which itoriginated, optionally coupled with the determination of other peptidesfrom that sample sample, the precursor protein to the degraded peptidecan be identified and/or quantified with respect to the sample fromwhich it originated. Because this method allows for the multiplexdetermination of a protein, or proteins, in more than one sample (i.e.from a sample mixture), it is a multiplex method.

[0101] In some embodiments, this invention pertains to a methodcomprising reacting each of two or more samples, each sample containingone or more reactive analytes, with a different labeling reagent of aset of labeling reagents wherein the different labeling reagents of theset each comprise the formula: RP-X-LK-Y-RG. Consequently, one or moreanalytes of each sample are labeled with the moiety “RP-X-LK-Y-” byreaction of a nucleophile or electrophile of the analyte with theelectrophilic or nucleophilic reactive group (RG), respectively, of thedifferent labeling reagents. The labeling process can produce two ormore differentially labeled samples each comprising one or more labeledanalytes. The labeling reagents of the set can be isomeric or isobaric.The reporter of each labeling reagent can be identified with, andtherefore used to identify, the sample from which each labeled analyteoriginated.

[0102] RG is a reactive group the characteristics of which have beenpreviously described. RP is a reporter moiety the characteristics ofwhich have been previously described. The gross mass of each reportercan be different for each reagent of the set. LK is a linker moiety thecharacteristics of which have been previously described. The gross massof the linker can compensate for the difference in gross mass betweenthe reporters for the different labeling reagents such that theaggregate gross mass of the reporter/linker combination is the same foreach reagent of the set. X is a bond between an atom of the reporter andan atom of the linker. Y is a bond between an atom of the linker and anatom of the reactive group (or after reaction with an analyte, Y is abond between the an atom of the linker and an atom of the analyte).Bonds X and Y fragment in at least a portion of the labeled analyteswhen subjected to dissociative energy levels in a mass spectrometer. Thecharacteristics of bonds X and Y have been previously described.

[0103] Once the analytes of each sample are labeled with the labelingreagent that is unique to that sample, the two or more differentiallylabeled samples, or a portion thereof, can be mixed to produce a samplemixture. Where quantitation is desired, the volume and/or quantity ofeach sample combined to produce the sample mixture can be recorded. Thevolume and/or quantity of each sample, relative to the total samplevolume and/or quantity of the sample mixture, can be used to determinethe ratio necessary for determining the amount (often expressed inconcentration and/or quantity) of an identified analyte in each samplefrom the analysis of the sample mixture. The sample mixture cantherefore comprise a complex mixture wherein relative amounts of thesame and/or different analytes can be identified and/or quantitated,either by relative quantitation of the amounts of analyte in each of thetwo or more samples or absolutely where a calibration standard is alsoadded to the sample mixture.

[0104] The mixture can then be subjected to spectrometry techniqueswherein a first mass analysis can be performed on the sample mixture, orfraction thereof, using a first mass analyzer. Ions of a particular massto charge ratio from the first mass analysis can then be selected. Theselected ions can then be subjected to dissociative energy levels (e.g.collision-induced dissociation (CID)) to thereby induce fragmentation ofthe selected ions. By subjecting the selected ions, of a particular massto charge ratio, of the labeled analytes to dissociative energy levels,both bonds X and Y can be fragmented in at least a portion of theselected ions. Fragmentation of both bonds X and Y can causefragmentation of the reporter/linker moiety as well as cause release thecharged or ionized reporter from the analyte. Ions subjected todissociative energy levels can also cause fragmentation of the analyteto thereby produce daughter fragment ions of the analyte. The ions(remaining selected ions, daughter fragment ions and charged or ionizedreporters), or a fraction thereof, can then be directed to a second massanalyzer.

[0105] In the second mass analyzer, a second mass analysis can beperformed on the selected ions, and the fragments thereof. The secondmass analysis can determine the gross mass (or m/z) and relative amountof each unique reporter that is present at the selected mass to chargeratio as well as the gross mass of the daughter fragment ions of atleast one reactive analyte of the sample mixture. For each analytepresent at the selected mass to charge ratio, the daughter fragment ionscan be used to identify the analyte or analytes present at the selectedmass to charge ratio. For example, this analysis can be done aspreviously described in the section entitled: “Analyte Determination ByComputer Assisted Database Analysis”.

[0106] In some embodiments, certain steps of the process can be repeatedone or more times. For example, in some embodiments, ions of a selectedmass to charge ratio from the first mass spectrometric analysis,different from any previously selected mass to charge ratio, can betreated to dissociative energy levels to thereby form ionized reportermoieties and ionized daughter fragment ions of at least some of theselected ions, as previously described. A second mass analysis of theselected ions, the ionized reporter moieties and the daughter fragmentions, or a fraction thereof, can be performed. The gross mass andrelative amount of each reporter moiety in the second mass analysis andthe gross mass of the daughter fragment ions can also be determined. Inthis way, the information can be made available for identifying andquantifying one or more additional analytes from the first massanalysis.

[0107] In some embodiments, the whole process can be repeated one ormore times. For example, it may be useful to repeat the process one ormore times where the sample mixture has been fractionated (e.g.separated by chromatography or electrophoresis). By repeating theprocess on each sample, it is possible to analyze all the entire samplemixture. It is contemplated that in some embodiments, the whole processwill be repeated one or more times and within each of these repeats,certain steps will also be repeated one or more times such as describedabove. In this way, the contents of sample mixture can be interrogatedand determined to the fullest possible extent.

[0108] Those of ordinary skill in the art of mass spectrometry willappreciate that the first and second mass analysis can be performed in atandem mass spectrometer. Instruments suitable for performing tandemmass analysis have been previously described herein. Although tandemmass spectrometers are preferred, single-stage mass spectrometers may beused. For example, analyte fragmentation may be induced by cone-voltagefragmentation, followed by mass analysis of the resulting fragmentsusing a single-stage quadrupole or time-of-flight mass spectrometer. Inother examples, analytes may be subjected to dissociative energy levelsusing a laser source and the resulting fragments recorded followingpost-source decay in time-of-flight or tandem time-of-flight (TOF-TOF)mass spectrometers.

[0109] According to the preceding disclosed multiplex methods, in someembodiments, bond X can be more or less prone to, or substantially equalto, fragmentation as compared with fragmentation of bonds of the analyte(e.g. an amide (peptide) bond in a peptide backbone). In someembodiments, bond Y can be more or less prone to fragmentation ascompared with fragmentation of bonds of the analyte (e.g. an amide(peptide) bond in a peptide backbone). In some embodiments, the linkerfor each reagent of the set is neutral in charge after the fragmentationof bonds X and Y (i.e. the linker fragments to produce a neutral loss ofmass and is therefore not observed in the MS/MS spectrum). In still someother embodiments, the position of bonds X and Y does not vary withinthe labeling reagents of a set, within the labeled analytes of a mixtureor within the labeling reagents of a kit. In yet some other embodiments,the reporter for each reagent of the set does not substantiallysub-fragment under conditions that are used to fragment the analyte(e.g. an amide (peptide) bond of a peptide backbone). In yet some otherembodiments, bond X is less prone to fragmentation as compared with bondY. In still some other embodiments, bond Y is less prone tofragmentation as compared with bond X. In still some other embodiments,bonds X and Y are of approximately the same lability or otherwise areselected such that fragmentation of one of bonds X or Y results in thefragmentation of the other of bonds X or Y. Other characteristics of thegroups that for the RP-X-LK-Y- moiety of labeled analytes havepreviously been described.

[0110] In some embodiments, the label of each isobarically labeledanalyte can be a 5, 6 or 7 membered heterocyclic ring comprising a ringnitrogen atom that is N-alkylated with a substituted or unsubstitutedacetic acid moiety to which the analyte is linked through the carbonylcarbon of the N-alkyl acetic acid moiety, wherein each different labelcan comprise one or more heavy atom isotopes. The heterocyclic ring canbe substituted or unsubstituted. The heterocyclic ring can be aliphaticor aromatic. Possible substituents of the heterocylic moiety includealkyl, alkoxy and aryl groups. The substituents can comprise protectedor unprotected groups, such as amine, hydroxyl or thiol groups, suitablefor linking the analyte to a support. The heterocyclic ring can compriseadditional heteroatoms such as one or more nitrogen, oxygen or sulfuratoms.

[0111] In some embodiments, labeled analytes in the sample mixture canbe isobars and each comprise the general formula:

[0112] wherein: Z is O, S, NH or NR¹; each J is the same or differentand is H, deuterium (D), R¹, OR¹, SR¹, NHR¹, N(R¹)₂, fluorine, chlorine,bromine or iodine; W is an atom or group that is located ortho, meta orpara to the ring nitrogen and is NH, N—R¹, N—R², P—R¹, P—R², O or S;each carbon of the heterocyclic ring has the formula CJ₂; each R¹ is thesame or different and is an alkyl group comprising one to eight carbonatoms which may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms; and R² is an amino alkyl, hydroxy alkyl, thio alkyl group or acleavable linker that cleavably links the reagent to a solid supportwherein the amino alkyl, hydroxy alkyl or thio alkyl group comprises oneto eight carbon atoms, which may optionally contain a heteroatom or asubstituted or unsubstituted aryl group, and wherein the carbon atoms ofthe alkyl and aryl groups independently comprise linked hydrogen,deuterium and/or fluorine atoms.

[0113] For example, the sample mixture can comprise one or moreisobarically labeled analytes of the general formula:

[0114] wherein isotopes of carbon 13 and oxygen 18 are used to balancethe gross mass between the morpholine reporter and the carbonyl linkerof the different labeling reagents.

[0115] Morpholine labeling reagents suitable to produce labeled analytesof this general structure can be prepared by numerous synthetic routes.For example, isotopically labeled or non-isotopically morpholinecompounds can be reacted with isotopically labeled or non-isotopicallylabeled bromoacetic acid compounds as described in Example 1. It shouldlikewise be apparent that a ring-substituted morpholine and/orsubstituted bromoacetic acid starting materials can also be selected andused by one of skill in the art without the exercise of undueexperimentation (with little or no change to the above describedprocedure or other procedures well-known in the art) to thereby producevarious different morpholine based labeling reagents, of differing heavyatom isotope content (i.e. isotopically coded), that can be used in thesets or kits of this invention.

[0116] Instead of morpholine, it is possible to choose a substituted orunsubstituted piperidine of desired isotopic distribution. Whenpiperidine is chosen, the isotopes D (deuterium) ¹³C or ¹⁵N can besubstituted for H, ¹²C and ¹⁴N, respectively, and used to alter thegross mass of the reagents of a set of labeling reagents in a mannersimilar to that illustrated for morpholine except that in the case ofpiperidine, ¹⁸O is not used in the ring atoms. An exemplary synthesis ofa piperidine, optionally using isotopically enriched starting materials,is described in Example 6.

[0117] The sample mixture can comprise one or more isobarically labeledanalytes of the formula:

[0118] wherein isotopes of carbon 13, oxygen 18 and nitrogen 15 are usedto balance the gross mass between the reporter and the carbonyl linkerof the different labeling reagents. Piperazine labeling reagentssuitable to produce labeled analytes of this general structure can beprepared by numerous synthetic routes. For example, heavy or lightpiperazine compounds can be reacted with heavy or light labeledbromoacetic acid compounds as described in Example 7. With reference toFIGS. 9A and 9B, a general schematic is shown for two differentsynthetic routes to isotopically enriched piperazines using readilyavailable heavy or light starting materials.

[0119] Specifically with reference to FIG. 9A, two equivalents of¹⁵N-labeled glycine 1 can be condensed to form the bis-isotopicallylabeled di-ketopiperazine 2 (the isotopic label is represented by the *in the Figure). The di-ketopiperazine can then be reduced to anisotopically labeled piperazine. The isotopically labeled piperazine canthen be reacted with bromoacetic acid and converted to an active ester 3as described in Example 7.

[0120] Specifically with reference to FIG. 9B, bis-¹⁵N-labeledethylenediamine 4 can be condensed with oxalic acid 5 to for thebis-isotopically labeled di-ketopiperazine 6 (the isotopic label isrepresented by the * in the Figure). The di-ketopiperazine can then bereduced to an isotopically labeled piperazine. The isotopically labeledpiperazine can then be reacted with bromoacetic acid and converted to anactive ester 3 as described in Example 7.

[0121] It should likewise be apparent that a ring-substituted piperazinecan be made using the above-described methods by merely choosingappropriately substituted starting materials. Where appropriate, asubstituted bromoacetic acid (either heavy or light) can likewise beused. By heavy we mean that the compound is isotopically enriched withone or more heave atom isotopes). By light we mean that it is notisotopically enriched. Accordingly, appropriately substituted startingmaterials can be selected to thereby produce various differentpiperazine based labeling reagents that can be used in the sets of thisinvention.

[0122] For example, the sample mixture can comprise one or moreisobarically labeled analytes of the formula:

[0123] wherein: isotopes of carbon 13, oxygen 18 and nitrogen 15 areused to balance the gross mass between the reporter and the carbonyllinker of the different labeling reagents and wherein; 1) each R¹ is thesame or different and is an alkyl group comprising one to eight carbonatoms which may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms; and 2) each K is independently selected as hydrogen or an aminoacid side chain. Substituted piperazine labeling reagents suitable toproduce labeled analytes of this general structure can be prepared bynumerous synthetic routes.

[0124] For example, with reference to FIG. 10, N-alkyl substitutedpiperazine reagents can be prepared in accordance with the illustratedprocedure. The tert-butyloxycarbonyl (t-boc) protected glycine 10 can becondensed with the ester (e.g. ethyl ester) of N-methyl-glycine 11 tothereby form the ester of the t-boc protected glycine-N-methyl-glycinedimer 12. The gly-gly dimer 12 can then be cyclized by removal of thet-boc protecting group followed by condensation to thereby form the acidsalt of the N-methyl-di-ketopiperazine 13. The acid salt of 13 can beneutralized and reduced to form the N-methyl-piperazine 14. TheN-methyl-piperazine 14, can then be reacted with bromoacetic acid 15 (orsubstituted versions thereof and converted to an active ester 16 asdescribed in Example 7.

[0125] It should be apparent that a ring-substituted piperazine can bemade using the above-described method by merely choosing an amino acidor N-methyl amino acid (or ester thereof other than glycine (e.g.alanine, phenylalanine, leucine, isoleucine, valine, asparagine, aparticacid, etc). It should likewise be apparent that the amino acids can beisotopically labeled in a manner suitable for preparing ring substitutedpiperazines having the desired distribution of isotopes necessary toprepare sets of isobaric labeling reagents.

[0126] N-alkyl substituted piperazine reagents can be prepared inaccordance by still another illustrated procedure. With reference toFIG. 11, glycine methyl ester 21 can be reacted with the ethyl ester ofbromoacetic acid 22 to form the diethyl iminodiacetate 23. The diesterof the diethyl iminodiacetate 23 can be converted to a di-acid chloride24 by treatment an appropriate reagent (e.g. thionyl chloride). Thedi-acid chloride 24 can then be reacted with, for example, an alkylamine (e.g. methyl amine) to form an N-alkyl-di-ketopiperazine 25. TheN-alkyl-di-ketopiperazine can then be reduced to form theN-alkyl-piperazine 26. The N-alkyl-piperazine can then be reacted withbromoacetic acid and converted to an active ester 27 as described inExample 7.

[0127] It should be apparent that a ring-substituted piperazine can bemade using the above-described method by merely choosing an ester of anamino acid other than glycine (e.g. alanine, phenylalanine, leucine,isoleucine, valine, asparagine, apartic acid, etc) or a substitutedversion of bromoacetic acid. It should likewise be apparent that theamino acids and bromoacetic acid (and its substituted derivatives) canbe isotopically labeled in a manner suitable for preparing ringsubstituted piperazines having the desired distribution of isotopesnecessary to prepare sets of isobaric labeling reagents. It should befurther apparent that choosing an alkyl diamine, hydroxyalkyl amine orthioalkylamine, or isotopically labeled version thereof, instead of analkyl amine can be used to produce the support bound labeling reagentsas described in more detail below.

[0128] In yet some other embodiments of the method, labeled analytes inthe sample mixture are isobars and each comprise the formula:

[0129] wherein: Z is O, S, NH or NR¹; each J is the same or differentand is selected from the group consisting of: H, deuterium (D), R¹, OR¹,SR¹, NHR¹, N(R¹)₂, fluorine, chlorine, bromine and iodine; each R¹ isthe same or different and is an alkyl group comprising one to eightcarbon atoms which may optionally contain a heteroatom or a substitutedor unsubstituted aryl group wherein the carbon atoms of the alkyl andaryl groups independently comprise linked hydrogen, deuterium and/orfluorine atoms.

[0130] For example, the sample mixture can comprise two or moreisobarically labeled analytes of the formula:

[0131] wherein isotopes of carbon 13 and oxygen 18 are used to balancethe gross mass between the reporter and the carbonyl linker of thedifferent labeling reagents. Substituted labeling reagents suitable toproduce labeled analytes of this general structure can be prepared bythe general process described in Example 8.

[0132] In still some other embodiments of this invention, each differentlabeling reagent of a set or kit of labeling reagents can be linked to asupport through a cleavable linker such that each different sample canbe reacted with a support carrying a different labeling reagent. In someembodiments, the supports can themselves be used for the labeling ofreactive analytes. In some embodiments, the labeling reagents can beremoved from the supports and then used, in some cases after subsequentprocessing (e.g. protection of reactive groups), for the labeling ofreactive analytes.

[0133] According to some embodiments, the analytes from a sample can bereacted with the solid support (each sample being reacted with adifferent solid support and therefore a different reporter) and theresin bound components of the sample that do not react with the reactivegroup can be optionally washed away. The labeled analyte or analytes canthen be removed from each solid support by treating the support underconditions that cleave the cleavable linker and thereby release thereporter/linker/analyte complex from the support. Each support can besimilarly treated under conditions that cleave the cleavable linker tothereby obtain two or more different samples, each sample comprising oneor more labeled analytes wherein the labeled analytes associated with aparticular sample can be identified and/or quantified by the uniquereporter linked thereto. The collected samples can then be mixed to forma sample mixture, as previously described.

[0134] For example, each different labeling reagent of the set used inthe previously described method can be a solid support of the formula:E-F-RP-X-LK-Y-RG, wherein; RG, X, Y, RP and LK have been describedpreviously. E is a solid support and F is a cleavable linker linked tothe solid support and cleavably linked to the reporter. Supports of thisgeneral formula can be prepared as described in Example 9.

[0135] In some embodiments, a set of support bound labeling reagents canbe based on labeled N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine derivatives. Both heavy and light piperazinederivatives can be prepared. The labeled N-(aminoalkyl), N-(thioalkyl)or N-(hydroxyalkyl)-piperazine derivatives can be formed, for example,by using the procedure illustrated in FIG. 11 starting with an alkyldiamine, thioalkyl amine or hydroxyalkyl amine as the N-alkyl amine (seethe discussion of FIG. 11, above). The alkyl diamine, thioalkyl amine orhydroxyalkyl amine can be heavy or light where appropriate for synthesisof a desired N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine derivative. The amino, hydroxyl or thiolgroup of the N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine derivatives can be protected as appropriate.When an alkyl diamine, thioalkylamine or hydroxyalkyl amine is used, thepiperazine can comprise an N-aminoalkyl, N-thioalkyl or N-hydroxyalkylmoiety wherein the amino, hydroxyl or thiol group of the moiety can bereacted with the cleavable linker on a support to thereby cleavably linkthe piperazine, prepared from the N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine derivative, to the support.

[0136] The support comprising a labeling reagent can be prepared by anyof several methods. In some embodiments, the amino, hydroxyl or thiolgroup of the N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine can be reacted with the cleavable linker ofa suitable support. The cleavable linker can be a “sterically hinderedcleavable linker” (See: Example 9). The piperazine can be reacted withisotopically labeled or non-isotopically labeled haloacetic acid(substituted or unsubstituted) depending on the nature of the labelingreagent desired for the set of labeling reagents. Thereafter thecarboxylic acid can be converted to an active ester. The active estercan be reacted with analytes of a sample to thereby label the analyteswith the labeling reagent of the support. Cleavage of the cleavablelinker will release the labeled analyte from the support. This processcan be repeated with an unique piperazine based labeling reagent for thepreparation of the different supports of a set of labeling supports.

[0137] In some embodiments, the N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine can be first reacted with isotopicallylabeled or non-isotopically labeled haloacefic acid (substituted orunsubstituted), or an ester thereof. Preferably, the amino, hydroxyl orthiol group of the N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine can be protected with a suitable protectingreagent (For a list of suitable protecting groups See: Green et al.,Protecting Groups In Organic Synthesis, Third Edition, John Wiley &Sons, Inc. New York, 1999). The unprotected amino, thiol or hydroxylgroup of the resulting bis-alkylated piperazine can then be reacted withthe cleavable linker of a suitable support. Thereafter the carboxylicacid can be converted to an active ester. If the haloacetic acidcompound was an ester, the ester can be saponified prior to conversionto an active ester. The active ester can be reacted with analytes of asample to thereby label the analytes with the labeling reagent of thesupport. Cleavage of the cleavable linker will release the labeledanalyte from the support. This process can be repeated with a uniquepiperazine based labeling reagent for the preparation of the differentsupports of a set of labeling supports.

[0138] Therefore, in some embodiments, the set of labeling reagents cancomprise one or more of the following support bound labeling reagents:

[0139] wherein RG, E and F have been previously described. According tothe method, G can be an amino alkyl, hydroxy alkyl or thio alkyl group,cleavably linked to the cleavable linker wherein the amino alkyl,hydroxy alkyl or thio alkyl group comprises one to eight carbon atoms,which may optionally contain a heteroatom or a substituted orunsubstituted aryl group, and wherein the carbon atoms of the alkyl andaryl groups independently comprise linked hydrogen, deuterium and/orfluorine atoms. Each carbon of the heterocyclic ring can have theformula CJ₂, wherein each J is the same or different and is selectedfrom the group consisting of H, deuterium (D), R¹, OR¹, SR¹, NHR¹,N(R¹)₂, fluorine, chlorine, bromine and iodine. Each R¹ can be the sameor different and is an alkyl group comprising one to eight carbon atomswhich may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms.

[0140] In some embodiments, the labeled analytes can be generated byfirst reacting the analyte with a support comprising the labelingreagent, cleavably linked to the support through a cleavable linker, andthen cleaving the labeled analyte from the support. Accordingly, asample mixture can comprise one or more isobarically labeled analytes ofthe formula:

[0141] wherein: G′ can be an amino alkyl, hydroxy alkyl or thio alkylgroup comprising one to eight carbon atoms which may optionally containa heteroatom or a substituted or unsubstituted aryl group wherein thecarbon atoms of the alkyl and aryl groups independently comprise linkedhydrogen and/or deuterium atoms. Each carbon of the heterocyclic ringcan have the formula CJ₂, wherein each J is the same or different and isselected from the group consisting of: H, deuterium (D), R¹, OR¹, SR¹,NHR¹, N(R¹)₂, fluorine, chlorine, bromine and iodine. Each R¹ can be thesame or different and is an alkyl group comprising one to eight carbonatoms which may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms. Here the alkyl amine group, hydroxy alkyl group or thio alkylgroup can be the moiety that was linked to the cleavable linker of thesolid support. The product of each cleavage reaction can be combined toproduce a sample mixture suitable for analysis of labeled analytes bythe methods described herein.

[0142] In some embodiments, methods of the invention can furthercomprise digesting each sample with at least one enzyme to partially, orfully, degrade components of the sample prior to performing the labelingof the analytes of the sample (Also see the above section entitled:“Sample Processing”). For example, the enzyme can be a protease (todegrade proteins and peptides) or a nuclease (to degrade nucleic acids).The enzymes may also be used together to thereby degrade samplecomponents. The enzyme can be a proteolytic enzyme such as trypsin,papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin orcarboxypeptidease C.

[0143] In some embodiments, methods can further comprise separating thesample mixture prior to performing the first mass analysis (Also see theabove section entitled: “Separation Of The Sample Mixture”). In thismanner the first mass analysis can be performed on only a fraction ofthe sample mixture. The separation can be performed by any separationsmethod, including by chromatography or by electrophoresis. For example,liquid chromatography/mass spectrometry (LC/MS) can be used to effectsuch a sample separation and mass analysis. Moreover, anychromatographic separation process suitable to separate the analytes ofinterest can be used. Non-limiting examples of suitable chromatographicand electrophoretic separations processes have been described herein.

[0144] In still other embodiments, the methods of the invention cancomprise both an enzyme treatment to degrade sample components and aseparations step.

[0145] As described previously, it is possible to determine the analyteassociated with the selected ions by analysis of the gross mass of thedaughter fragment ions. One such method of determination is described inthe section entitled: “Analyte Determination By Computer AssistedDatabase Analysis”.

[0146] Once the analyte has been determined, information regarding thegross mass and relative amount of each reporter moiety in the secondmass analysis and the gross mass of daughter fragment ions provides thebasis to determine other information about the sample mixture. Theamount of reporter can be determined by peak intensity in the massspectrum. In some embodiments, the amount of reporter can be determinedby analysis of the peak height or peak width of the reporter (signatureion) signal obtained using the mass spectrometer. Because each samplecan be labeled with a different labeling reagent and each labelingreagent can comprise a unique reporter that can be correlated with aparticular sample, determination of the different reporters in thesecond mass analysis identifies the sample from which the ions of theselected analyte originated. Where multiple reporters are found (e.g.according to the multiplex methods of the invention), the relativeamount of each reporter can be determined with respect to the otherreporters. Because the relative amount of each reporter determined inthe second mass analysis correlates with the relative amount of ananalyte in the sample mixture, the relative amount (often expressed asconcentration and/or quantity) of the analyte in each sample combined toform the sample mixture can be determined. As appropriate, a correctionof peak intensity associated with the reporters can be performed fornaturally occurring, or artificially created, isotopic abundance, aspreviously discussed. More specifically, where the volume and/orquantity of each sample that is combined to the sample mixture is known,the relative amount (often expressed as concentration and/or quantity)of the analyte in each sample can be calculated based upon the relativeamount of each reporter determined in the second mass analysis.

[0147] This analysis can be repeated one or more times on selected ionsof a different mass to charge ratio to thereby obtain the relativeamount of one or more additional analytes in each sample combined toform the sample mixture. As appropriate, a correction of peak intensityassociated with the reporters can be performed for naturally occurring,or artificially created, isotopic abundance.

[0148] Alternatively, where a calibration standard comprising a uniquereporter linked to an analyte, having the selected mass to charge ratio,has been added to the sample mixture in a known amount (often expressedas a concentration and/or quantity), the amount of the unique reporterassociated with the calibration standard can be used to determine theabsolute amount (often expressed as a concentration and/or quantity) ofthe analyte in each of the samples combined to form the sample mixture.This is possible because the amount of analyte associated with thereporter for the calibration standard is known and the relative amountsof all other reporters can be determined for the labeled analyteassociated with the selected ions. Since the relative amount ofreporter, determined for each of the unique reporters (including thereporter for the calibration standard), is proportional to the amount ofthe analyte associated with each sample combined to form the samplemixture, the absolute amount (often expressed as a concentration and/orquantity) of the analyte in each of the samples can be determined basedupon a ratio calculated with respect to the formulation used to producethe sample mixture. As appropriate, a correction of peak intensityassociated with the reporters can be performed for naturally occurring,or artificially created, isotopic abundance.

[0149] This analysis can be repeated one or more times on selected ionsof a different mass to charge ratio to thereby obtain the absoluteamount of one or more additional analytes in each sample combined toform the sample mixture. As appropriate, a correction of peak intensityassociated with the reporters can be performed for naturally occurring,or artificially created, isotopic abundance.

[0150] In some embodiments, the methods can be practiced with digestionand/or separation steps. In some embodiments, the steps of the methods,with or without the digestion and/or separation steps, can be repeatedone or more times to thereby identify and/or quantify one or more otheranalytes in a sample or one or more analytes in each of the two or moresamples (including samples labeled with support bound labelingreagents). Depending of whether or not a calibration standard is presentin the sample mixture for a particular analyte, the quantitation can berelative to the other labeled analytes, or it can be absolute. Such ananalysis method can be particularly useful for proteomic analysis ofmultiplex samples of a complex nature, especially where a preliminaryseparation of the labeled analytes (e.g. liquid chromatography orelectrophoretic separation) precedes the first mass analysis.

[0151] In some embodiments, the analytes can be peptides in a sample orsample mixture. Analysis of the peptides in a sample, or sample mixture,can be used to determine the amount (often expressed as a concentrationand/or quantity) of identifiable proteins in the sample or samplemixture wherein proteins in one or more samples can be degraded prior tothe first mass analysis. Moreover, the information from differentsamples can be compared for the purpose of making determinations, suchas for the comparison of the effect on the amount of the protein incells that are incubated with differing concentrations of a substancethat may affect cell growth. Other, non-limiting examples may includecomparison of the expressed protein components of diseased and healthytissue or cell cultures. This may encompass comparison of expressedprotein levels in cells, tissues or biological fluids followinginfection with an infective agent such as a bacteria or virus or otherdisease states such as cancer. In other examples, changes in proteinconcentration over time (time-course) studies may be undertaken toexamine the effect of drug treatment on the expressed protein componentof cells or tissues. In still other examples, the information fromdifferent samples taken over time may be used to detect and monitor theconcentration of specific proteins in tissues, organs or biologicalfluids as a result of disease (e.g. cancer) or infection.

[0152] In some embodiments, the analyte can be a nucleic acid fragmentin a sample or sample mixture. The information on the nucleic acidfragments can be used to determine the amount (often expressed as aconcentration and/or quantity) of identifiable nucleic acid molecules inthe sample or sample mixture wherein the sample was degraded prior tothe first mass analysis. Moreover, the information from the differentsamples can be compared for the purpose of making determinations asdescribed above.

[0153] B. Mixtures

[0154] In some embodiments, this invention pertains to mixtures (i.e.sample mixtures). The mixtures can comprise at least two differentiallylabeled analytes, wherein each of the two-labeled analytes can originatefrom a different sample and comprise the formula: RP-X-LK-Y-Analyte. Foreach different label, some of the labeled analytes of the mixture can bethe same and some of the labeled analytes can be different. The atoms,moieties or bonds, X, Y, RP and LK have been previously described andtheir characteristics disclosed. The mixture can be formed by mixingall, or a part, of the product of two or more labeling reactions whereineach labeling reaction uses a different labeling reagent of the generalformula: RP-X-LK-Y-RG, wherein atoms, moieties or bonds X, Y, RP, LK RGhave been previously described and their characteristics disclosed. Thelabeling reagents can be isotopically coded isomeric or isobariclabeling reagents. The unique reporter of each different labelingreagent can indicate from which labeling reaction each of the two ormore labeled analytes is derived. The labeling reagents can be isomericor isobaric. Hence, two or more of the labeled analytes of a mixture canbe isomeric or isobaric. The mixture can be the sample mixture asdisclosed in any of the above-described methods. Characteristics of thelabeling reagents and labeled analytes associated with those methodshave been previously discussed.

[0155] The analytes of the mixture can be peptides. The analytes of themixture can be proteins. The analytes of the mixture can be peptides andproteins. The analytes of the mixture can be nucleic acid molecules. Theanalytes of the mixture can be carbohydrates. The analytes of themixture can be lipids. The analytes of the mixture can be steroids. Theanalytes of the mixture can be small molecules of less than 1500daltons. The analytes of the mixture comprise two or more analyte types.The analyte types can, for example, be selected from peptides, proteins,nucleic acids carbohydrates, lipids, steroids and/or small molecules ofless than 1500 daltons.

[0156] In some embodiments, the label of each isobarically labeledanalyte can be a 5, 6 or 7 membered heterocyclic ring comprising a ringnitrogen atom that is N-alkylated with a substituted or unsubstitutedacetic acid moiety to which the analyte is linked through the carbonylcarbon of the N-alkyl acetic acid moiety, wherein each different labelcomprises one or more heavy atom isotopes. The heterocyclic ring can besubstituted or unsubstituted. The heterocyclic ring can be aliphatic oraromatic. Possible substituents of the heterocylic moiety include alkyl,alkoxy and aryl groups. The substituents can comprise protected orunprotected groups, such as amine, hydroxyl or thiol groups, suitablefor linking the analyte to a support. The heterocyclic ring can compriseadditional heteroatoms such as one or more nitrogen, oxygen or sulfuratoms.

[0157] In some embodiments, the labeled analytes of the mixture areisobars and each comprise the formula:

[0158] wherein Z, J and W have been previously described and theircharacteristics disclosed. For example, the sample mixture can compriseone or more isobarically labeled analytes of the formula:

[0159] wherein isotopes of carbon 13 and oxygen 18 are used to balancethe gross mass between the morpholine reporter and the carbonyl linkerof the different labeling reagents.

[0160] In some embodiments, the sample mixture can comprise one or moreisobarically labeled analytes of the formula:

[0161] wherein isotopes of carbon 13, oxygen 18 and nitrogen 15 are usedto balance the gross mass between the reporter and the carbonyl linkerof the different labeling reagents. In some embodiments, the samplemixture can comprise one or more isobarically labeled analytes of theformula:

[0162] wherein: isotopes of carbon 13, oxygen 18 and nitrogen 15 areused to balance the gross mass between the reporter and the carbonyllinker of the different labeling reagents and wherein; 1) each R¹ is thesame or different and is an alkyl group comprising one to eight carbonatoms which may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms; and 2) each K is independently selected as hydrogen or an aminoacid side chain.

[0163] In some embodiments, the labeled analytes of the mixture areisobars and each comprise the formula:

[0164] wherein: Z, J and R¹ have been previously described and theircharacteristics disclosed. For example, the sample mixture can compriseone or more isobarically labeled analytes of the formula:

[0165] wherein isotopes of carbon 13 and oxygen 18 are used to balancethe gross mass between the reporter and the carbonyl linker of thedifferent labeling reagents.

[0166] In other embodiments, the labeled analytes can be generated byfirst reacting the analyte with a support comprising the labelingreagent, cleavably linked to the support through a cleavable linker, andthen cleaving the labeled analyte from the support. For example thelabeled analytes of the mixture can be one or more isobars comprisingthe general formula:

[0167] wherein: G′ has been previously described and its characteristicsdisclosed.

[0168] C. Kits

[0169] In some embodiments, this invention pertains to kits. The kitscan comprise a set of two or more labeling reagents of the formula:RP-X-LK-Y-RG and one or more reagents, containers, enzymes, buffersand/or instructions. The atoms, moieties or bonds X, Y, RP, LK RG havebeen previously described and their characteristics disclosed. Thelabeling reagents of a kit can be isomeric or isobaric. Other propertiesof the labeling reagents of the kits have likewise been disclosed. Forexample, the kits can be useful for the multiplex analysis of one ormore analytes in the same sample, or in two or more different samples.

[0170] In some embodiments, the label of each isobarically labeledanalyte can be a 5, 6 or 7 membered heterocyclic ring comprising a ringnitrogen atom that is N-alkylated with a substituted or unsubstitutedacetic acid moiety to which the analyte is linked through the carbonylcarbon of the N-alkyl acetic acid moiety, wherein each different labelcomprises one or more heavy atom isotopes. The heterocyclic ring can besubstituted or unsubstituted. The heterocyclic ring can be aliphatic oraromatic. Possible substituents of the heterocylic moiety include alkyl,alkoxy and aryl groups. The substituents can comprise protected orunprotected groups, such as amine, hydroxyl or thiol groups, suitablefor linking the analyte to a support. The heterocyclic ring can compriseadditional heteroatoms such as one or more nitrogen, oxygen or sulfuratoms.

[0171] In some embodiments, the different reagents of a kit are isobarsand each comprise the formula:

[0172] wherein RG, Z, J and W have been previously described and theircharacteristics disclosed. For example, the reagents of a kit cancomprise one or more isobarically labeled reagents of the formula:

[0173] wherein RG is the reactive group and isotopes of carbon 13 andoxygen 18 are used to balance the gross mass between the morpholinereporter and the carbonyl linker of the different labeling reagents.

[0174] In some embodiments, the kit can comprise one or moreisobarically labeled reagents of the formula:

[0175] wherein RG is the reactive group and isotopes of carbon 13,oxygen 18 and nitrogen 15 are used to balance the gross mass between thereporter and the carbonyl linker of the different labeling reagents. Insome embodiments, the reagents of a kit can comprise one or moreisobarically labeled reagents of the formula:

[0176] wherein: isotopes of carbon 13, oxygen 18 and nitrogen 15 areused to balance the gross mass between the reporter and the carbonyllinker of the different labeling reagents and wherein; 1) each R¹ is thesame or different and is an alkyl group comprising one to eight carbonatoms which may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms; and 2) each K is independently selected as hydrogen or an aminoacid side chain. In yet other embodiments, the labeled analytes of thekit are isobars and each comprises the formula:

[0177] wherein: RG Z, J and R¹ have been previously described and theircharacteristics disclosed. For example, the reagents of a kit cancomprise one or more isobarically labeled analytes of the formula:

[0178] wherein RG has been previously described and disclosed andisotopes of carbon 13 and oxygen 18 are used to balance the gross massbetween the reporter and the carbonyl linker of the different labelingreagents.

[0179] In some embodiments, this invention pertains to kits comprisingone or more sets of supports, each support comprising a differentlabeling reagent, cleavably linked to the support through a cleavablelinker. For example, the cleavable linker can be chemically orphotolytically cleavable. The supports can be reacted with differentsamples thereby labeling the analytes of a sample with the samereporter/linker, and analytes of different samples with differentreporter/linker combinations. Supports of a set that can be used inembodiments of this invention have the general formula:E-F-G-RP-X-LK-Y-RG, wherein E, F, G, RP, X, LK, Y and RG have beenpreviously defined herein and their characteristics disclosed. Eachdifferent support of the set can comprise a unique reporter.

[0180] For example the supports of a kit can comprise two or more of thereagent supports of the formula:

[0181] wherein: E, F, G and RG have been previously described and theircharacteristics disclosed.

[0182] In some embodiments, the kit comprises a proteolytic enzyme. Theproteolytic enzyme can be trypsin, papain, pepsin, ArgC, LysC, V8protease, AspN, pronase, chymotrypsin or carboxypeptidease C. In someembodiments, the kit can comprise instructions for using the labelingreagents to differentially label the analytes of different samples.

[0183] D. Compositions

[0184] In some embodiments, this invention pertains to compositions thatcan be used as labeling reagents. The compositions can be labelingreagents of the formula: RP-X-LK-Y-RG,

[0185] wherein the atoms, moieties or bonds X, Y, RP, LK RG have beenpreviously described and their characteristics disclosed. The labelingreagents can be isomeric or isobaric. Other properties of the labelingreagents have likewise been disclosed. For example, the labelingreagents can be useful for the multiplex analysis of one or moreanalytes in the same sample, or in two or more different samples.

[0186] The labeling reagents can be isotopically enriched (coded) withat least one heavy atom isotope. The labeling reagents can beisotopically enriched to comprise two or more heavy atom isotopes. Thelabeling reagents can be isotopically enriched to comprise three or moreheavy atom isotopes. The labeling reagents can be isotopically enrichedto comprise four or more heavy atom isotopes. In some embodiments, atleast one heavy atom isotope is incorporated into a carbonyl orthiocarbonyl group of the labeling reagent and at least one other heavyatom isotope is incorporated into the reporter group of the labelingreagent.

[0187] Each incorporated heavy atom isotope can be present in at least80 percent isotopic purity. Each incorporated heavy atom isotope can bepresent in at least 93 percent isotopic purity. Each incorporated heavyatom isotope can be present in at least 96 percent isotopic purity.

[0188] The labeling reagents comprise a reporter group that contains afixed charge or that is ionizable. The reporter group therefore caninclude basic or acidic moieties that are easily ionized. In someembodiments, the reporter can be a morpholine, piperidine or piperazinecompound. In some embodiments, the reporter can be a carboxylic acid,sulfonic acid or phosphoric acid group containing compound. Accordingly,is some embodiments, the labeling reagents can be isolated in their saltform. For example, piperazine containing labeling reagents can beobtained as a mono-TFA salt, a mono-HCl salt, a bis-TFA salt or abis-HCl salt. The number of counterions present in the labeling reagentcan depend in the number of acidic and/or basic groups present in thelabeling reagent.

[0189] In some embodiments, the labeling reagents can comprise acarbonyl or thiocarbonyl linker. Labeling reagents comprising a carbonylor thiocarbonyl linker can be used in active ester form for the labelingof analytes. In an active ester, an alcohol group forms a leaving group(LG). In some embodiments, the alcohol (LG) of the active ester can havethe formula:

[0190] wherein X is O or S. The active ester can be anN-hydroxysuccinimidyl ester.

[0191] In some embodiments, the active ester compound can be a 5, 6 or 7membered heterocyclic ring comprising a ring nitrogen atom that isN-alkylated with a substituted or unsubstituted acetic acid moiety towhich the alcohol moiety of the active ester is linked through thecarbonyl carbon of the N-alkyl acetic acid moiety, wherein the compoundis isotopically enriched with one or more heavy atom isotopes. Theheterocyclic ring of the active ester can be substituted with one ormore substituents. The one or more substituents can be alkyl, alkoxy oraryl groups. The one or more substituents can be alkylamine,alkylhydroxy or alkylthio groups. The one or more substituents can beprotected or unprotected amine groups, hydroxyl groups or thiol groups.The heterocyclic ring can be aliphatic. The heterocyclic ring can bearomatic. The heterocyclic ring can comprise one or more additionalnitrogen, oxygen or sulfur atoms.

[0192] In some embodiments, the active ester compound can be anN-substituted morpholine acetic acid active ester compound of theformula:

[0193] or a salt thereof, wherein; LG is the leaving group of an activeester; X is O or S; each Z is independently hydrogen, deuterium,fluorine, chlorine, bromine, iodine, an amino acid side chain or astraight chain or branched C1-C6 alkyl group that may optionally containa substituted or unsubstituted aryl group wherein the carbon atoms ofthe alkyl and aryl groups each independently comprise linked hydrogen,deuterium or fluorine atoms. In some embodiments, Z independently can behydrogen, deuterium, fluorine, chlorine, bromine or iodine. In someembodiments, Z independently can be hydrogen, methyl or methoxy. In someembodiments, X is ¹⁶O or ¹⁸O. The nitrogen atom of the morpholine ringcan be ¹⁴N or ¹⁵N. In some embodiments, the active ester is a compoundcomprising the formula:

[0194] wherein each C* is independently ¹²C or ¹³C; LG is the leavinggroup of an active ester; X is O or S; and each Z is independentlyhydrogen, deuterium, fluorine, chlorine, bromine, iodine, an amino acidside chain or a straight chain or branched C1-C6 alkyl group that mayoptionally contain a substituted or unsubstituted aryl group wherein thecarbon atoms of the alkyl and aryl groups each independently compriselinked hydrogen, deuterium or fluorine atoms.

[0195] In some embodiments, the active ester compound can be anN-substituted piperidine acetic acid active ester compound of theformula:

[0196] or a salt thereof, wherein; LG is the leaving group of an activeester; X is O or S; each Z is independently hydrogen, deuterium,fluorine, chlorine, bromine, iodine, an amino acid side chain or astraight chain or branched C1-C6 alkyl group that may optionally containa substituted or unsubstituted aryl group wherein the carbon atoms ofthe alkyl and aryl groups each independently comprise linked hydrogen,deuterium or fluorine atoms. In some embodiments, Z independently can behydrogen, deuterium, fluorine, chlorine, bromine or iodine. In someembodiments, Z independently can be hydrogen, methyl or methoxy. In someembodiments X is ¹⁶O or ¹⁸O. The nitrogen atom of the piperidine ringcan be ¹⁴N or ¹⁵N. In some embodiments, the active ester is a compoundcomprising the formula:

[0197] wherein each C* is independently ¹²C or ¹³C; LG is the leavinggroup of an active ester; X is O or S; and each Z is independentlyhydrogen, deuterium, fluorine, chlorine, bromine, iodine, an amino acidside chain or a straight chain or branched C1-C6 alkyl group that mayoptionally contain a substituted or unsubstituted aryl group wherein thecarbon atoms of the alkyl and aryl groups each independently compriselinked hydrogen, deuterium or fluorine atoms.

[0198] In some embodiments, the active ester compound can be anN-substituted piperidine acetic acid active ester compound of theformula:

[0199] or a salt thereof, wherein; LG is the leaving group of an activeester; X is O or S; Pg is an amine protecting group; and each Z isindependently hydrogen, deuterium, fluorine, chlorine, bromine, iodine,an amino acid side chain or a straight chain or branched C1-C6 alkylgroup that may optionally contain a substituted or unsubstituted arylgroup wherein the carbon atoms of the alkyl and aryl groups eachindependently comprise linked hydrogen, deuterium or fluorine atoms. Insome embodiments, Z independently can be hydrogen, deuterium, fluorine,chlorine, bromine or iodine. In some embodiments, Z independently can behydrogen, methyl or methoxy. In some embodiments X is ¹⁶O or ¹⁸O. Insome embodiments, each nitrogen atom of the piperazine ring is ¹⁴N or¹⁵N. In some embodiments, the active ester is a compound comprising theformula:

[0200] wherein each C* is independently ¹²C or ¹³C; LG is the leavinggroup of an active ester; X is O or S; Pg is an amine protecting groupand each Z is independently hydrogen, deuterium, fluorine, chlorine,bromine, iodine, an amino acid side chain or a straight chain orbranched C1-C6 alkyl group that may optionally contain a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups each independently comprise linked hydrogen, deuterium orfluorine atoms.

[0201] Having described embodiments of the invention, it will now becomeapparent to one of skill in the art that other embodiments incorporatingthe concepts may be used. It is felt, therefore, that these embodimentsshould not be limited to disclosed embodiments but rather should belimited only by the spirit and scope of the invention.

EXAMPLES

[0202] This invention is now illustrated by the following examples thatare not intended to be limiting in any way.

Example 1 Synthesis of Morpholine Acetic Acid

[0203] Bromoacetic acid (2 g, 14.4 mole) was dissolved intetrahydrofuran (50 mL) and added dropwise to a stirred solution ofmorpholine (3.76 g, 43.2 mole) in tetrahydrofuran (THF, 20 mL). Thesolution was stirred at room temperature for three days. The white solid(4.17 g) was filtered, washed with THF (100 mL), and recrystallised fromhot ethanol (EtOH), Yield: 2.59 g: IR:1740 cm-1. For the two differentisobaric versions of morpholine acetic acid, either bromoacetic-1-¹³Cacid (Aldrich PN 27,933-1) or bromoacetic-2-¹³C acid (Aldrich PN27,935-8) was substituted for bromoacetic acid.

Example 2 Synthesis of Morpholine Acetic Acid N-HydroxysucciniimideEster

[0204] Dimethylformamide (dry, 1.75 g, 0.024M) was dissolved intetrahydrofuran (dry, 30 mLs). This solution was added dropwise to astirred solution of thionyl chloride (2.85 g, 0.024M) dissolved intetrahydrofuran(dry, 20 mLs) and cooled in an ice bath. After completeaddition and 30 minutes on ice, the ice bath was removed and solidN-hydroxysuccinimide (2 g, 0.017 M) was added (which completelydissolved) immediately followed by solid pre-powdered morpholine aceticacid [or -1-¹³C or -2-¹³C morpholine acetic acid] (3.64 g, 0.016M). Themorpholine acetic acid dissolved slowly giving a homogeneous solutionthat rapidly became cloudy. The reaction was left vigorously stirringover night at room temperature. The white solid was washed withtetrahydrofuran and dried under vacuum, weight 3.65 g (67%), IR spectrum1828.0 cm-1, 1790.0 cm-1, 1736.0 cm-1.

Example 3 Analyte Determination and Relative Quantitation in Two Samples

[0205] 100 pmole amounts of freeze-dried Glu-Fibrinopeptide B (Sigma)were reacted with 200 μl of freshly-made 2% w/v solutions of either I orII (See: FIG. 1A for structure and Examples 1 & 2 for preparation) inice-cold 0.5M MOPS buffer (pH 7.8 with NaOH) for 30 minutes on ice. Thereaction was terminated by the addition of TFA to 0.5% v/v finalconcentration. The modified peptides were then mixed in variouspre-determined proportions to approximately cover the range 1:10 to 10:1of the differentially labeled peptides. Each peptide mixture wasindividually purified by reverse-phase de-salting using a Millipore C18Zip-Tip. Excess reagent and buffer do not retain on the reverse-phasepackings, and were thus efficiently removed prior to MS analysis. Themixtures (0.5 μl) were then spotted onto a MALDI target plate,over-spotted with 0.5 μl of 1% w/v α-cyano cinnamic acid in 50% aqueousacetonitrile and each sample was analyzed using a MALDI source fitted toa QTOF analyzer.

[0206]FIG. 2 is an expansion plot of the MS spectrum obtained from the1:1 mix of Glu-fibrinopeptide as modified with reagents I and II. Thepeak at m/z 1699 represents the N-terminally modified mass ofGlu-fibrinopeptide, and as expected, there is no observable differencein m/z of the two different forms of the peptide (See: FIGS. 1A(III) and1A(IV). The modified peptides are isobaric. The isotopic clusterobserved for the peak is exactly as expected for a single species.

[0207] The singly-charged precursor ion of m/z 1699 was then selectedfor fragmentation by low energy CID (collision offset of approximately−70V), yielding the MS/MS spectrum found in FIG. 3. As expected, theobserved ion series was predominantly of types b- and y-. All these ionsappeared as single species, with no indication that they comprised a 1:1mixture of the differentially-labeled peptide species. For example, anexpansion of the prominent y-ion at m/z 1056.5 is shown in the expansionplot as FIG. 4 and the prominent b-ion at m/z 886.3 is shown in theexpansion plot as FIG. 5. TABLE 1 Observed Predicted 0.13 0.125 0.170.166 0.2 0.25 0.46 0.5 1.03 1 2.15 2 4.16 4 6.3 6 7.9 8

[0208] Close examination of the spectrum at about 100 m/z (FIG. 6),however, reveals the presence of both species VII and VIII (FIG. 1B),which are the fragmentation products of species V and VI (FIG. 1B),respectively. No peaks are observed at m/z 128.1, thereby indicatingthat species V and VI are not stable enough to be observed. In thisexample, therefore, it may be that fragmentation of the amide bondbetween the carbonyl group and the amino-terminal amino acid of thepeptide (e.g. bond Y) induced subsequent fragmentation of thereporter/linker moiety (bond X) and loss of the carbonyl moiety asneutral CO. Peak integration was performed using the instrumentationprovided with the instrument. Following compensation for the naturallyoccurring second C-13 isotopic contribution of approximately 6 percent,the measured relative ratio of VIII/VII (101/100) was 1.03 (expectedvalue 1.00). Table 1 shows actual versus observed ratios for additionalexperimental mixtures prepared (ratio expressed as intensity m/z 101/m/z100), with correction for the naturally occurring second C-13contribution. This data is also represented graphically in FIG. 7. Thereis excellent agreement between observed and predicted values, with meanerror <10%.

Example 4 Proteomic Analysis

[0209] In practice, a representative proteomic analysis can be performedas follows. Total cellular protein extracts for comparison (e.g. samplesA and B) are separately digested with trypsin, or another proteolyticenzyme. The resulting peptide mixtures are separately reacted withdifferent isomeric of isobaric labeling reagents (for example, compoundsI and II) to give complete modification of N-terminal and lysine aminesof the peptides. For example, sample A can be reacted with compound Iand sample B can be reacted with compound II. Each of the samplescontaining modified peptides/proteins are then be mixed together beforechromatographic separation (often using multi-dimensional HPLC) andanalyzed by MS and MS/MS techniques. The labeling can be performed witha single label treatment (no prior blocking of lysine groups with asecond reagent required) as the groups are isobaric.

[0210] The mixture of labeled proteins/peptides is thenchromatographically separated and the eluent, or fractions thereof,analyzed by mass spectrometry as described in Example 3, above.Effective sensitivity may also be significantly increased usingtriple-quadruople or Q-trap mass spectrometers, where the m/z region of100 and 101 is monitored in precursor-ion mode. The relative ratios ofthe two “signature” peaks are directly correlated with the ratio of eachpeptide/protein analyte of interest in each of samples A and B. As usedherein, the “signature” peaks are the peaks for the reporter.

Example 5 Analyte Determination and Quantitation Relative to An InternalStandard

[0211] Total cellular protein extracts for comparison (e.g. samples Aand B) are separately digested with trypsin. The resulting peptidemixtures are separately reacted with X and XI (FIG. 8) to givesubstantially complete modification of N-terminal and lysine amines asdescribed above. For example, sample A peptides are reacted with X andsample B peptides are reacted with XI. Known amounts or each of samplesA and B, containing substantially modified peptides, are then mixedtogether. To the combined mixture of A and B is now added, in accuratelydetermined amount, a set (one or more) of synthetic peptide(s) thatcorrespond exactly in amino acid sequence and/or post-translationalmodification (e.g. phosphorylation) to peptide(s) that may be present inthe mixture of samples A and B, and where the synthetic peptide(s) arelabeled with another member of set of isobaric labeling reagents (e.g.compounds XII or XIII, see: FIG. 8). The combined mixture of peptidesfrom sample A, sample B and synthetic internal standard peptides canoptionally be subjected to chromatographic separation, for example bymulti-dimensional HPLC, or electrophoretic separation and then analyzedby MS and MS/MS techniques as described previously. All equivalentlabeled peptides from sample A, B and synthetic counterparts ofidentical sequence are isobaric and have substantially identicalchromatographic properties. By “substantially identical chromatographicproperties” we mean that there is very little, if any, separation of thedifferentially labeled but otherwise identical peptides. Following MS/MSanalysis, the absolute concentration of peptides from sample A and B maybe accurately determined by comparison of the relative intensity of thereporters for X (sample A) and for XI (sample B) with respect to theintensity of the reporter (the “signature peak”) resulting from thestandard peptide labeled with the additional member of the isobaric set(e.g. XII or XIII).

[0212] Although the foregoing is a description of two samples (i.e.Samples A and B), this process could be extended in many practical ways.For example, there may be many samples that are analyzed simultaneouslyprovided there is a large enough set of labeling reagents.

[0213] There could be a double (or more where there are more samples tobe analyzed) internal standard (e.g. sample A peptides may be ‘spiked’with synthetic peptides labeled with reagent XII and sample B peptidesmay be spiked with synthetic peptides labeled with reagent XIII (ofknown absolute concentration)). When all are combined, separated andanalyzed as described above, Sample A peptides can be quantitatedrelative to the signature peak for compound XII and sample B peptidescan be quantitated relative to the signature peak for compound XIII.

Example 6 Exemplary Synthesis of Piperidine Acetic AcidN-hydroxysuccinimide Ester

[0214] Bromoacetic acid is dissolved in tetrahydrofuran (or anothersuitable non-nucleophilic solvent) and added dropwise to a stirredsolution containing an excess of piperidine in tetrahydrofuran (THF, oranother suitable non-nucleophilic solvent). The solution is stirred atroom temperature for one to three days. The solid is filtered, washedwith THF (or another suitable non-nucleophilic solvent), and optionallyrecrystallised. For the two different isobaric versions of piperidineacetic acid, either bromoacetic-1-¹³C acid (Aldrich PN 27,933-1) orbromoacetic-2-¹³C acid (Aldrich PN 27,935-8) can be substituted forbromoacetic acid. Isomer substituted piperidine can be prepared fromsuitable starting material or it can be obtained, on a custom orderbasis, from sources such as Cambridge Isotope Laboratories or Isotec.

[0215] To convert the acetic acid derivatives to active esters, such asan N-hydroxysuccinimidyl ester, dimethylformamide (DMF) is dissolved intetrahydrofuran (or another suitable non-nucleophilic solvent). Thissolution is added dropwise to a stirred solution of an equal molaramount of thionyl chloride (based upon the molar quantity of DMF)dissolved in tetrahydrofuran (or another suitable non-nucleophilicsolvent) and cooled in an ice bath. After complete addition and 30minutes on ice, the ice bath is removed and solid N-hydroxysuccinimideis added immediately followed by piperidine acetic acid (or -1-¹³C or-2-¹³C piperidine acetic acid). The reaction is left vigorously stirringover night at room temperature. The product piperidine acetic acidN-hydroxysuccinimide ester is then isolated from the reaction mixturepossibly by mere filtration. Recrystallization and/or chromatography canoptionally be used to purify the crude product.

Example 7 Exemplary Synthesis of Piperazine Acetic AcidN-hydroxysuccinimide Ester

[0216] A solution containing two equivalents of piperazine dissolved intetrahydrofuran (THF) is added dropwise to a solution containing oneequivalent of bromoacetic acid (as compared with the amount ofpiperazine) dissolved in tetrahydrofuran. The two solutions should be asconcentrated as is practical. The resulting reaction solution is stirredat room temperature for one to three days. The solid is filtered, washedwith THF, and optionally recrystallised. For the two different isobaricversions of piperidine acetic acid, either bromoacetic-1-¹³C acid(Aldrich PN 27,933-1) or bromoacetic-2-¹³C acid (Aldrich PN 27,935-8)can be substituted for bromoacetic acid.

[0217] To convert the acetic acid derivatives to active esters, such asan N-hydroxysuccinimidyl ester, dry dimethylformamide (DMF, 1.75 g,0.024M) can be dissolved in tetrahydrofuran. This solution can be addeddropwise to a stirred solution of an equal molar amount of thionylchloride (based upon the molar quantity of DMF) dissolved intetrahydrofuran and cooled in an ice bath. After complete addition and30 minutes on ice, the ice bath can be removed and solidN-hydroxysuccinimide added immediately followed by piperazine aceticacid (or -1-¹³C or -2-¹³C piperidine acetic acid). The reaction can beleft vigorously stirring over night at room temperature. The productpiperazine acetic acid N-hydroxysuccinimide ester can then be isolatedfrom the reaction mixture possibly by mere filtration. Recrystallizationor chromatography can then be used to purify the crude product.

Example 8 Exemplary Synthesis of N,N′-(2-methoxyethyl)-glycine ActiveEster (Copied from U.S. Pat. No. 6,326,479

[0218] To 1.1 mole of bis(2-methoxyethyl)amine (Aldrich Chemical) wasadded dropwise 500 mmol of tert-butyl chloroacetate (Aldrich Chemical).The reaction was allowed to stir for three days and was then worked up.To the final reaction contents was added 250 mL of dichloromethane (DCM)and 200 mL of water. To this stirring solution was added portionwise,300 mmol of solid potassium carbonate (K₂CO₃). After complete mixing,the layers were separated. The DCM layer was washed once with a volumeof water, dried (Na₂SO₄), filtered and evaporated to yield 66.3 g of avery thin yellow oil. This crude product was Kugelrohr distilled at 60°C. (200-500 μM Hg) to yield 58.9 g of a clear colorless oil (238 mmol;95%).

[0219] To the purified (stirring)N,N′-(2-methoxyethyl)-glycine-tert-butyl ester was slowly added 12.1 mLof concentrated hydrochloric acid. The reaction was allowed to stirovernight and then the byproducts (e.g. water, HCl, isobutylene) wereremoved by vacuum evaporation. ¹H-MNR analysis indicated the t-butylester was hydrolyzed but it appeared that there was water and HCl stillpresent. The crude product was co-evaporated 2× from acetonitrile (ACN)but water and HCl were still present. To eliminate impurities, a 4.4 galiquot was removed from the crude product and Kugelrohr distilled at135-155° C. (100-200 μM Hg with rapidly dropping pressure afterdistillation began). Yield 4.2 g (18.4 mmol; 95% recovery of thick,clear, colorless oil). The distilled product did not contain any wateror HCl.

[0220] The active ester (e.g. N-hydroxysuccinimidyl ester) of anysuitable isotopically labelled substituted or unsubstitutedN,N′-(2-methoxyethyl)-glycine can then be prepared by methods known inthe art, such as those described herein.

Example 9 Exemplary Method for Preparing a Solid Support ComprisingLabelling/Tagging Reagents

[0221] A commercially available peptide synthesis resin comprising a“sterically hindered cleavable linker” is reacted with at least two-foldexcess of an aminoalkyl piperazine (e.g. 1-(2-aminoethyl)piperazine,Aldrich P/N A5,520-9; isomeric versions can be made by the processillustrated in FIG. 11 in combination with the description in thespecification). By “sterically hindered cleavable linker” we mean thatthe linker comprises a secondary or tertiary atom that forms thecovalent cleavable bond between the solid support and the atom or groupreacted with the cleavable linker. Non-limiting examples of stericallyhindered solid supports include: Trityl chloride resin (trityl-Cl,Novabiochem, P/N 01-64-0074), 2-Chlorotrityl chloride resin(Novabiochem, P/N 01-64-0021), DHPP (Bachem, P/N Q-1755), MBHA (AppliedBiosystems P/N 400377), 4-methyltrityl chloride resin (Novabiochem, P/N01-64-0075), 4-methoxytrityl chloride resin (Novabiochem, P/N01-64-0076), Hydroxy-(2-chorophnyl)methyl-PS (Novabiochem, P/N01-64-0345), Rink Acid Resin (Novabiochem P/Ns 01-64-0380, 01-64-0202),NovaSyn TGT alcohol resin (Novabiochem, P/N 01-64-0074). Excess reagentsare then removed by washing the support. The secondary amine of thesupport bound piperazine is then reacted with an excess of bromoaceticacid in the presence of a tertiary amine such as triethylamine. Excessreagents are then removed by washing the support. Depending on themethod to be used to make an active ester of the carboxylic acid (e.g.whether or not a salt of the carboxylic acid is required for the activeester synthesis), the wash can be selected to have a pH that is adjustedto protonate the support bound carboxylic acid group of thebis-alkylated piperazine. The carboxylic acid group of the support boundpiperazine is then converted to an active ester (e.g.N-hydroxysuccinimidyl ester) using procedures known in the art for theproduction of acid esters of a carboxylic acid, such as those describedabove. The resulting solid support can thereafter be used to labelanalytes of a sample (e.g. peptides) having nucleophilic functionalgroups. The labeled analytes can then be released from the support asdescribed by the manufacturer's product instructions. The product ofeach cleavage reaction can then be combined to form a sample mixture.

We claim:
 1. An active ester compound that is a 5, 6 or 7 memberedheterocyclic ring comprising a ring nitrogen atom that is N-alkylatedwith a substituted or unsubstituted acetic acid moiety to which thealcohol moiety of the active ester is linked through the carbonyl carbonof the N-alkyl acetic acid moiety, wherein the compound is isotopicallyenriched with one or more heavy atom isotopes.
 2. The compound of claim1, wherein the compound is isotopically enriched with three or moreheavy atom isotopes.
 3. The compound of claim 1, wherein theheterocyclic ring is substituted with one or more substituents.
 4. Thecompound of claim 3, wherein the one or more substituents are alkyl,alkoxy or aryl groups.
 5. The compound of claim 4, wherein the one ormore substituents are protected or unprotected amine groups, hydroxylgroups or thiol groups.
 6. The compound of claim 1, wherein theheterocyclic ring is aliphatic.
 7. The compound of claim 1, wherein theheterocyclic ring is aromatic.
 8. The compound of claim 1, wherein theheterocyclic ring comprises one or more additional nitrogen, oxygen orsulfur atoms.
 9. The compound of claim 1, wherein active ester is anN-hydroxysuccinimide ester.
 10. The compound of claim 1, wherein thecompound is a salt.
 11. The compound of claim 1, wherein the compound isa mono-TFA salt, a mono-HCl salt, a bis-TFA salt or a bis-HCl salt. 12.The compound of claim 1, wherein each incorporated heavy atom isotope ispresent in at least 80 percent isotopic purity.
 13. The compound ofclaim 1, wherein each incorporated heavy atom isotope is present in atleast 93 percent isotopic purity.
 14. The compound of claim 1, whereineach incorporated heavy atom isotope is present in at least 96 percentisotopic purity.
 15. An N-substituted morpholine acetic acid activeester compound of the formula:

or a salt thereof, wherein; LG is the leaving group of an active ester;X is O or S; each Z is independently hydrogen, deuterium, fluorine,chlorine, bromine, iodine, an amino acid side chain or a straight chainor branched C1-C6 alkyl group that may optionally contain a substitutedor unsubstituted aryl group wherein the carbon atoms of the alkyl andaryl groups each independently comprise linked hydrogen, deuterium orfluorine atoms; and wherein the N-substituted morpholine acetic acidactive ester is isotopically enriched with one or more heavy atomisotopes.
 16. The compound of claim 15, wherein the N-substitutedmorpholine acetic acid active ester is isotopically enriched with threeor more heavy atom isotopes.
 17. The compound of claim 15, wherein LGis:

and wherein X is O or S.
 18. The compound of claim 15, wherein LG isN-hydroxysuccinimide.
 19. The compound of claim 15, wherein each Z isindependently hydrogen, deuterium, fluorine, chlorine, bromine oriodine.
 20. The compound of claim 15, wherein each Z is independentlyhydrogen, methyl or methoxy.
 21. The compound of claim 15, wherein X is¹⁶O or ¹⁸O.
 22. The compound of claim 15, wherein the nitrogen atom ofthe morpholine ring is ¹⁴N or ¹⁵N.
 23. The compound of claim 15, of theformula:

wherein; each C* is independently ¹²C or ¹³C; LG is the leaving group ofan active ester; X is O or S; and each Z is independently hydrogen,deuterium, fluorine, chlorine, bromine, iodine, an amino acid side chainor a straight chain or branched C1-C6 alkyl group that may optionallycontain a substituted or unsubstituted aryl group wherein the carbonatoms of the alkyl and aryl groups each independently comprise linkedhydrogen, deuterium or fluorine atoms.
 24. The compound of claim 15,wherein the compound is a mono-TFA salt or a mono-HCl salt.
 25. Thecompound of claim 15, wherein each incorporated heavy atom isotope ispresent in at least 80 percent isotopic purity.
 26. The compound ofclaim 15, wherein each incorporated heavy atom isotope is present in atleast 93 percent or isotopic purity.
 27. The compound of claim 15,wherein each incorporated heavy atom isotope is present in at least 96percent or isotopic purity.
 28. An N-substituted piperidine acetic acidactive ester compound of the formula:

or a salt thereof, wherein; LG is the leaving group of an active ester;X is O or S; each Z is independently hydrogen, deuterium, fluorine,chlorine, bromine, iodine, an amino acid side chain or a straight chainor branched C1-C6 alkyl group that may optionally contain a substitutedor unsubstituted aryl group wherein the carbon atoms of the alkyl andaryl groups each independently comprise linked hydrogen, deuterium orfluorine atoms; and wherein the N-substituted piperidine acetic acidactive ester is isotopically enriched with one or more heavy atomisotopes.
 29. The compound of claim 28, wherein the N-substitutedpiperidine acetic acid active ester is isotopically enriched with threeor more heavy atom isotopes.
 30. The compound of claim 28, wherein LGis:

and wherein X is O or S.
 31. The compound of claim 28, wherein LG isN-hydroxysuccinimide.
 32. The compound of claim 28, wherein each Z isindependently hydrogen, deuterium, fluorine, chlorine, bromine oriodine.
 33. The compound of claim 28, wherein each Z is independentlyhydrogen, methyl or methoxy.
 34. The compound of claim 28, wherein X is¹⁶O or ¹⁸O.
 35. The compound of claim 28, wherein the nitrogen atom ofthe piperidine ring is ¹⁴N or ¹⁵N.
 36. The compound of claim 28, of theformula:

wherein; each C* is independently ¹²C or ¹³C; LG is the leaving group ofan active ester; X is O or S; and each Z is independently hydrogen,deuterium, fluorine, chlorine, bromine, iodine, an amino acid side chainor a straight chain or branched C1-C6 alkyl group that may optionallycontain a substituted or unsubstituted aryl group wherein the carbonatoms of the alkyl and aryl groups each independently comprise linkedhydrogen, deuterium or fluorine atoms.
 37. The compound of claim 28,wherein the compound is a mono-TFA salt or a mono-HCl salt.
 38. Thecompound of claim 28, wherein each incorporated heavy atom isotope ispresent in at least 80 percent isotopic purity.
 39. The compound ofclaim 28, wherein each incorporated heavy atom isotope is present in atleast 93 percent or isotopic purity.
 40. The compound of claim 28,wherein each incorporated heavy atom isotope is present in at least 96percent or isotopic purity.
 41. An N-substituted piperazine acetic acidactive ester compound of the formula:

or a salt thereof, wherein; LG is the leaving group of an active ester;X is O or S; Pg is an amine-protecting group; each Z is independentlyhydrogen, deuterium, fluorine, chlorine, bromine, iodine, an amino acidside chain or a straight chain or branched C1-C6 alkyl group that mayoptionally contain a substituted or unsubstituted aryl group wherein thecarbon atoms of the alkyl and aryl groups each independently compriselinked hydrogen, deuterium or fluorine atoms; and wherein theN-substituted piperazine acetic acid active ester is isotopicallyenriched with one or more heavy atom isotopes.
 42. The compound of claim41, wherein the N-substituted piperazine acetic acid active ester isisotopically enriched with three or more heavy atom isotopes
 43. Thecompound of claim 41, wherein LG is:

and wherein X is O or S.
 44. The compound of claim 41, wherein LG isN-hydroxysuccinimide.
 45. The compound of claim 41, wherein each Z isindependently hydrogen, deuterium, fluorine, chlorine, bromine oriodine.
 46. The compound of claim 41, wherein each Z is independentlyhydrogen, methyl or methoxy.
 47. The compound of claim 41, wherein X is¹⁶O or ¹⁸O.
 48. The compound of claim 41, wherein each nitrogen atom ofthe piperazine ring is ¹⁴N or ¹⁵N.
 49. The compound of claim 41, of theformula:

wherein, each C* is independently ¹²C or ¹³C; LG is the leaving group ofan active ester; X is O or S; Pg is an amine protecting group; and eachZ is independently hydrogen, deuterium, fluorine, chlorine, bromine,iodine, an amino acid side chain or a straight chain or branched C1-C6alkyl group that may optionally contain a substituted or unsubstitutedaryl group wherein the carbon atoms of the alkyl and aryl groups eachindependently comprise linked hydrogen, deuterium or fluorine atoms. 50.The compound of claim 41, wherein the compound is a mono-TFA salt, amono-HCl salt, a bis-TFA salt or a bis-HCl salt
 51. The compound ofclaim 41, wherein each incorporated heavy atom isotope is present in atleast 80 percent isotopic purity.
 52. The compound of claim 41, whereineach incorporated heavy atom isotope is present in at least 93 percentor isotopic purity.
 53. The compound of claim 41, wherein eachincorporated heavy atom isotope is present in at least 96 percent orisotopic purity.
 54. A kit comprising: a) a set of two or more reagentssuitable for the labeling of analytes, each reagent of the setcomprising the formula: RP-X-LK-Y-RG  or a salt thereof wherein; i) RGis a reactive group that is an electrophile and that is capable ofreacting with one or more of the reactive analytes of the sample; ii) RPis a reporter moiety that comprises a fixed charge or that is ionizable,wherein the gross mass of each reporter is different for each reagent ofthe set; iii) LK is a linker moiety that links the reactive group andthe reporter group, wherein: a) the mass of the linker compensates forthe difference in gross mass between the reporters for the differentlabeling reagents of the set such that the aggregate gross mass of thereporter and linker combination is the same for each reagent of the set;and b) the linker comprises at least one heavy atom isotope and has theformula:

wherein R¹ is the same or different and is an alkyl group comprising oneto eight carbon atoms which may optionally contain a heteroatom or asubstituted or unsubstituted aryl group wherein the carbon atoms of thealkyl and aryl groups independently comprise linked hydrogen, deuteriumand/or fluorine atoms; iv) X is a bond between an atom of the reporterand an atom of the linker; v) Y is a bond between an atom of the linkerand an atom of the reactive group, wherein, once the labeling reagent isreacted with the reactive analyte, bond Y links the linker to theanalyte; and b) one or more reagents, containers, enzymes, buffers orinstructions.
 55. The kit of claim 54, wherein the kit comprises aproteolytic enzyme.
 56. The kit of claim 54, wherein the reactive groupof each reagent of the set is an active ester.
 57. The kit of claim 56,wherein the alcohol moiety of the active ester is a group of theformula:

wherein X is O or S.
 58. The kit of claim 56, wherein the active esteris an N-hydroxysuccinimide ester.
 59. The kit of claim 54, wherein thereporter is a substituted or unsubstituted morpholine, piperidine orpiperazine.
 60. The kit of claim 54, wherein the reporter comprises acarboxylic acid, sulfonic acid or phosphoric acid group.
 61. The kit ofclaim 54, wherein the linker is a carbonyl or thiocarbonyl group. 62.The kit of claim 54, wherein each reagent of the set is independentlylinked to a solid support through a cleavable linker.
 63. The kit ofclaim 54, wherein all reagents of the set are isomeric.
 64. The kit ofclaim 54, wherein all reagents of the set are isobaric.
 65. The kit ofclaim 64, wherein all reagents of the set comprise the formula:

wherein; a) RG is a reactive group that is an electrophile; b) Z is O,S, NH or NR¹; c) each J is the same or different and is selected fromthe group consisting of: H, deuterium (D), R¹, OR¹, SR¹, NHR¹, N(R¹)₂,fluorine, chlorine, bromine and iodine; d) W is an atom or group that islocated ortho, meta or para to the ring nitrogen and is selected fromthe group consisting of: NH, N—R², P—R², O or S; and e) each carbon ofthe heterocyclic ring has the formula CJ₂; f) each R¹ is the same ordifferent and is an alkyl group comprising one to eight carbon atomswhich may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen deuterium and/or fluorineatoms; and g) R² is an amino alkyl, hydroxy alkyl, thio alkyl group or acleavable linker that cleavably links the reagent to a solid supportwherein the amino alkyl, hydroxy alkyl or thio alkyl group comprises oneto eight carbon atoms, which may optionally contain a heteroatom or asubstituted or unsubstituted aryl group, and wherein the carbon atoms ofthe alkyl and aryl groups independently comprise linked hydrogen,deuterium and/or fluorine atoms.
 66. The kit of claim 65, wherein theset comprises one or more of the following four reagents:

wherein RG is the reactive group.
 67. The kit of claim 65, wherein theset comprises one or more of the following four reagents:

wherein RG is the reactive group.
 68. The kit of claim 65, wherein theset comprises one or more of the following four support bound reagents:

wherein: a) RG is the reactive group; b) E is a solid support; c) F is acleavable linker linked to the solid support; d) G is an amino alkyl,hydroxy alkyl or thio alkyl group, cleavably linked to the cleavablelinker wherein the amino alkyl, hydroxy alkyl or thio alkyl groupcomprises one to eight carbon atoms, which may optionally contain aheteroatom or a substituted or unsubstituted aryl group, and wherein thecarbon atoms of the alkyl and aryl groups independently comprise linkedhydrogen, deuterium and/or fluorine atoms; e) each carbon of theheterocyclic ring has the formula CJ₂; and wherein each J is the same ordifferent and is selected from the group consisting of: H, deuterium(D), R¹, OR¹, SR¹, NHR¹, N(R¹)₂, fluorine, chlorine, bromine and iodine;and f) each R¹ is the same or different and is an alkyl group comprisingone to eight carbon atoms which may optionally contain a heteroatom or asubstituted or unsubstituted aryl group wherein the carbon atoms of thealkyl and aryl groups independently comprise linked hydrogen, deuteriumand/or fluorine atoms.
 69. The kit of claim 65, wherein all reagents ofthe set comprise the formula:

wherein: a) RG is a reactive group that is a nucleophile orelectrophile; b) Z is O, S, NH or NR¹; c) each J is the same ordifferent and is selected from the group consisting of: H, deuterium(D), R¹, OR¹, SR¹, NHR¹, N(R¹)₂, fluorine, chlorine, bromine and iodine;and d) each R¹ is the same or different and is an alkyl group comprisingone to eight carbon atoms which may optionally contain a heteroatom or asubstituted or unsubstituted aryl group wherein the carbon atoms of thealkyl and aryl groups independently comprise linked hydrogen deuteriumand/or fluorine atoms.
 70. The kit of claim 65, wherein the setcomprises one or more of the following four reagents:

wherein RG is the reactive group.